Mammalian interleukin-4

ABSTRACT

Mammalian proteins and muteins thereof, designated interleukin-4s (IL-4s), are provided which exhibit both B cell growth factor activity and T cell growth factor activity. Compounds of the invention include native human and murine IL-4s, muteins thereof, and nucleic acids which are effectively homologous to disclosed cDNAs, and/or which are capable of coding for mammalian IL-4s and their muteins.

CROSS REFERENCE TO RELATED APPLICATIONS

This is a continuation-in-part of copending U.S. patent application Ser.No. 881,553 filed July 3, 1986; now abandoned, which is acontinuation-in-part of copending application Ser. No. 843,958 filedMar. 25, 1986; which is a continuation-in-part of copending applicationSer. No. 799,668 filed Nov. 19, 1985; now abandoned, all of saidcopending applications being incorporated herein by reference.

BACKGROUND OF THE INVENTION

The present invention relates generally to protein and mutein factors ofthe mammalian immune system and to nucleic acids coding therefor. Moreparticularly, the invention relates to protein and mutein factors (alongwith their encoding nucleic acids) which exhibit both T cell growthfactor activity and B cell growth factor activity.

Recombinant DNA technology refers generally to the technique ofintegrating genetic information from a donor source into vectors forsubsequent processing, such as through introduction into a host, wherebythe transferred genetic information is copied and/or expressed in thenew environment. Commonly, the genetic information exists in the form ofcomplementary DNA (cDNA) derived from messenger RNA (mRNA) coding for adesired protein product. The carrier is frequently a plasmid having thecapacity to incorporate cDNA for later replication in a host and, insome cases, actually to control expression of the cDNA and therebydirect synthesis of the encoded product in the host.

For some time, it has been known that the mammalian immune response isbased on a series of complex cellular interactions, called the "immunenetwork." Recent research has provided new insights into the innerworkings of this network. While it remains clear that much of theresponse does, in fact, revolve around the network-like interactions oflymphocytes, macrophages, granulocytes and other cells, immunologistsnow generally hold the opinion that soluble proteins (e.g., theso-called "lymphokines" or "monokines") play a critical role incontrolling these cellular interactions. Thus, there is considerableinterest in the isolation, characterization, and mechanisms of action ofcell modulatory factors, an understanding of which should yieldsignificant breakthroughs in the diagnosis and therapy of numerousdisease states.

Lymphokines apparently mediate cellular activities in a variety of ways.They have been shown to support the proliferation, growth anddifferentiation of the pluripotential hematopoietic stem cells into thevast number of progenitors composing the diverse cellular lineagesresponsible for the immune response. These lineages often respond in adifferent manner when lymphokines are used in conjunction with otheragents.

Cell lineages that are especially important to the immune responseinclude two classes of lymphocytes: B-cells, which can produce andsecrete immunoglobulins (proteins with the capability of recognizing andbinding to foreign matter to effect its removal), and T-cells of varioussubsets that secrete lymphokines and induce or suppress the B-cells andsome of the other cells (including other T-cells) making up the immunenetwork.

Another important cell lineage is the mast cell (which has not beenpositively identified in all mammalian species)--a granule-containingconnective tissue cell located proximal to capillaries throughout thebody, with especially high concentrations in the lungs, skin, andgastrointestinal and genitourinary tracts. Mast cells play a centralrole in allergy-related disorders, particularly anaphylaxis as follows:when selected antigens crosslink one class of immunoglobulins bound toreceptors on the mast cell surface, the mast cell degranulates andreleases the mediators (e.g., histamine, serotonin, heparin,prostaglandins, etc.) which cause allergic reactions, e.g., anaphylaxis.

Research to better understand (and thus potentially treattherapeutically) various immune disorders has been hampered by thegeneral inability to maintain cells of the immune system in vitro.Immunologists have discovered that culturing these cells can beaccomplished through the use of T-cell and other cell supernatants,which contain various growth factors, such as some of the lymphokines.

The detection, isolation and purification of these factors is extremelydifficult, being frequently complicated by the complexity of thesupernatants they are typically located in, the divergencies andcross-overs of activities of the various components in the mixtures, thesensitivity (or lack thereof) of the assays utilized to ascertain thefactors' properties, the frequent similarity in the range of molecularweights and other characteristics of the factors, and the very lowconcentration of the factors in their natural setting.

As more lymphokines become available, primarily through molecularcloning, interest has heightened in finding clinical applications forthem. Because of physiological similarities to hormones (e.g., solublefactors, growth mediators, action via cell receptors), potential uses oflymphokines have been analogized to the current uses of hormones, e.g.Dexter, Nature, Vol. 321, pg. 198 (1986). One hope is that the levels oflymphokines in a patient can be manipulated directly or indirectly tobring about a beneficial immune response, e.g. suppression in the caseof inflammation, allergy, or tissue rejection, or stimulation orpotentiation in the case of infection or malignant growth. Otherpotential clinical uses of lymphokines include maintaining and expandingin vitro populations of certain immune system cells of one person foreventual reintroduction into the same or another person for a beneficialeffect. For example, investigations are currently underway to determinewhether populations of lymphokine-activated killer T cells of a patientcan be expanded outside his or her body then reinjected to bring aboutan enhanced antitumor response. Another potential clinical use oflymphokines, particularly colony stimulating factors, such asgranulocyte-macrophage colony stimulating factor (GM-CSF), and factorswhich enhance their activities, is stimulating blood cell generation,for example, in pre-or post-chemotherapy or radiation therapy againsttumors, in treatment of myeloid hypoplasias, or in treatment ofneutrophil deficiency syndromes, Dexter, Nature, Vol. 321, pg. 198(1986). Another area where such factors would be useful is in bonemarrow transplant therapy, which is being used increasingly to treataplastic anemia and certain leukemias.

There are two properties of lymphokines that have important consequencesfor such clinical applications: Individual lymphokines are frequentlypleiotropic. And the biological effects of one lymphokine can usually bemodulated by at least one other lymphokine, either by inhibition or bypotentiation. For example, tumor necrosis factor, which synergizes withgamma-interferon, stimulates interleukin-1 (IL-1) production and canactivate the phagocytic activity of neutrophils. IL-1, a proteinproduced by activated macrophages, mediates a wide range of biologicalactivities, including stimulation of thymocyte proliferation viainduction of interleukin-2 (IL-2) release, stimulation of B-lymphocytematuration and proliferation, fibroblast growth factor activity andinduction of acute-phase protein synthesis by hepatocytes. IL-1 has alsobeen reported to stimulate prostaglandin and collagenase release fromsynovial cells, and to be identical to endogenous pyrogen, Krampschmidt,J. Leuk. Biol., Vol. 36, pgs. 341-355 (1984).

Interleukin-2, formerly referred to as T-cell growth factor is alymphokine which is produced by lectin-or antigen-activated T cells. Thereported biological activities of IL-2 include stimulation of thelong-term in vitro growth of activated T-cell clones, enhancement ofthymocyte mitogenesis, and induction of cytotoxic T-cell reactivity andplaque-forming cell responses in cultures of nude mouse spleen cells. Inaddition, like interferons (IFNs), IL-2 has been shown to augmentnatural killer cell activity, suggesting a potential use in thetreatment of neoplastic diseases, Henney et al., Nature, Vol, 291, pgs.335-338 (1981). Some success has been reported in such therapy, e.g.Lotze and Rosenberg, "Treatment of Tumor Patients with Purified HumanInterleukin-2," pgs,. 711-719, in Sorg et al., Eds. Cellular andMolecular Biology of Lymphokines (Academic Press, Inc., New York, 1985);and Rosenberg and Lotze, "Cancer Immunotherapy Using Interleukin-2 andInterleukin-2 Activated Lymphocytes, "Ann. Rev. Immunol., Vol. 4, pgs.681-709 (1986). However, IL-2 toxicity has limited the dosages which canbe delivered to cancer patients for taking advantage of theseproperties, Lotze and Rosenberg, pgs. 711-719; and Welte et al., pgs.755-759, in Sorg et al. Eds. (cited above).

Metcalf, D., The Hematopoietic Colony Stimulating Factors, (Elsevier,Amsterdam, 1984), provides an overview of research concerninglymphokines and various growth factors involved in the mammalian immuneresponse. Yung, Y.-P., et al., J. Immunol. Vol. 127 pg. 794 (1981),describe the partial purification of the protein of approximately 35 kdexhibiting mast cell growth factor (MCGF) activity and its separationfrom interleukin-2 (IL-2), also known as T-cell growth factor (TCGF).Nabel, G., et al., Nature, Vol. 291, pg. 332 (1981) report an MCGFexhibiting a molecular weight of about 45 kd and a pI of about 6.0.Clark-Lewis, I. and Schrader, J., J. Immunol., Vol. 127, pg. 1941(1981), describe a protein having mast cell like growth factor activitythat exhibits a molecular weight of about 29 kd in phosphate-bufferedsaline and about 23 kd in 6M guanadine hydrochloride, with a pI ofbetween about 4-8 but of about 6-8 after neuraminidase treatment. MurineIL-2 and interleukins-3 (IL-3) have been partially characterizedbiochemically by Gillis, S., et al., J. Immunol., Vol. 124, pgs.1954-1962 (1980), and Ihle, J., et al., J. Immunol., Vol. 129, pgs.2431-2436 (1982), respectively, with IL-2 having an apparent molecularweight (probably as a dimer) of about 30-35 kd and IL-3 having amolecular weight of about 28 kd. Human IL-2 apparently has a molecularweight of about 15 kd and is described by Gillis, S., et al., Immu.Rev., Vol. 63, pgs. 167-209 (1982). Comparison between IL-3 and MCGFactivities of T-cell supernatants have been reported by Yung Y. andMoore, M., Contemp. Top. Mol. Immunol., Vol. 10, pgs. 147-179 (1985),and Rennick, D., et al., J. Immunol., Vol. 134, pgs. 910-919 (1985).

An extensive literature exists concerning the regulation of B-cellgrowth and differentiation by soluble factors, e.g. for reviews seeHoward and Paul, Ann. Rev. Immunol., Vol. 1, pgs. 307-333 (1983); Howardet al., Immunol. Rev., 1984, No. 78, pgs. 185-210; Kishimoto et al.,Immunol. Rev., 1984, No. 78, pgs. 97-118; and Kishimoto, Ann Rev.Immunol., Vol. 3, pgs. 133-157 (1985). Some confusion has existed overthe nomenclature used for labeling the various factors because ofdifferences in source materials, difficulties in purification, anddifferences in the assays used to define their biological activities.Consensus in regard to nomenclature apparently has been reached in somecases, Paul, Immunology Today, Vol. 4, pg. 322 (1983); and Paul,Molecular Immunol., Vol. 21, pg. 343 (1984). B-cell growth factor (BCGF)activity is characterized by a capacity to cause DNA synthesis in Bcells co-stimulated by exposure to anti-IgM, or like antigens. It isbelieved that interleukin-1 (IL-1) is also required for BCGF activity tobe manifested, at least when the assay is conducted with low densitiesof B cells. Alternative assays for human BCGF have been described, e.g.Maizel et al, Proc. Natl. Acad. Sci., Vol. 80, pgs. 5047-5051 (1983)(support of long-term growth of human B cells in culture). The activityassociated with the former assay has also been labelled B cellstimulatory factor-1 (BSF-1) activity and BCGF I, to distinguish it fromsimilar and/or related activities. In particular, an activity designatedBCGF II has been described. It is characterized by a capacity to causeDNA synthesis in mitogen stimulated B cells or in transformed B celllines. Mitogens associated with BCGF II activity include dextransulfate, lipopolysaccharide, and Staphylococcus extracts. BCGF Iregisters no response in these assays. In humans it is believed thatBCGF II is a molecule having a molecular weight of about 50 kilodaltons(kD), and that it acts synergistically with BCGF I (i.e. BSF-1) inpromoting B cell proliferation in an immune response, Yoshizaka et al.,J. Immunol., Vol. 130, pgs. 1241-1246 (1983). Howard et al., J. Exp.Med., Vol. 155, pgs. 914-923 ( 1982) were the first to show theexistence of a murine BCGF (later to be called variously BCGF I, BSF-1,or IgG₁ induction factor) distinct from interleukin-2. Similarobservations were reported almost simultaneously for a human system byYoshizaki et al., J. Immunol., Vol. 128, pgs. 1296-1301 (1982); andlater by Okada et al., J. Exp. Med., Vol. 157, pgs. 583-590 (1983).

Biochemical and biological characterization of molecules exhibitingBCGF, or BSF-1, activity has progressed steadily since these initialdiscoveries. Maizel et al., Proc. Natl. Acad. Sci., Vol. 79, pgs.5998-6002 (1982) , have reported a trypsin-sensitive human BCGF having amolecular weight of 12-13 kD and an isoelectric point (pI) of about6.3-6.6 . Farrar et al., J. Immunol., vol. 131, pgs. 1838-1842 (1983)reported partial purification of a heterogeneous murine BCGF havingmolecular weights of 11 and 15 kD by SDS-PAGE and pIs of 6.4-8.7. Oharaand Paul, in Nature, Vol. 315, pgs. 333-336 (1985) describe a monoclonalantibody specific for murine BSF-1, and molecular weights for BSF-1 of14 kD and 19-20 kD with pI of 6.7 Butler et al., J. Immunol., Vol. 133,pgs. 251-255 (1984), report a human BCGF having a molecular weight of18-20 kD and a pI of 6.3-6.6. Rubin et al., Proc. Natl. Acad. Sci., vol82, pgs. 2935-2939 (1985) report that pre-incubation of resting B cellswith BSF-1 prior to exposure to anti-IgM antibodies increses cellvolume, and later speeds entry to S phase upon exposure to anti-IgMantibodies. Vitetta et al, J. Exp. Med., Vol. 162, pgs. 1726-1731(1985), describe partial purification of murine BSF-1 by reverse phaseHPLC of serum free supernatants of EL-4 cells. SDS-PAGE indicated aprotein of about 20-22kD. Ohara et al., J. Immunol., Vol. 135, pgs.2518-2523 (1985) also report partial purification of murine BSF-1 by asimilar procedure, and report the factor to be a protein of about18-21.7 kD. Sideras et al., in Eur. J. Immunol., Vol. 15, pgs. 586-593,and 593-598 (1985), report partial purification of a murine IgG₁-inducing factor, that is a BSF-1, and report the factor to be a proteinof about 20 kD having pIs of 7.2-7.4 and 6.2-6.4, and Smith and Rennick,In Proc. Natl. Acad. Sci., Vol. 83, pgs. 1857-1861 (1986), report theseparation of a factor from IL-2 and IL-3 which exhibits T cell growthfactor activity and mast cell growth factor activity. Later, Noma etal., Nature, Vol. 319, pgs. 640-646 (1986), cloned and sequenced anucleic acid coding for the Sideras et al. factor, and Lee et al., Proc.Natl. Acad. Sci., Vol. 83, pgs. 2061-2065 (1986) cloned and sequenced anucleic acid coding for the Smith and Rennick factor. More recently,Gabstein et al., J. Exp. Med., vol. 163, pgs. 1405-1414 (1986), reportpurifying and sequencing murine BSF-1.

Milanese, et al, in Science, Vol. 231, pgs. 1118-1122 (1986), report alymphokine unrelated to BSF-1 which they provisionally designate IL-4A.Their IL-4A is a 10-12 kD protein secreted from helper T cells aftercross linking of T3-Ti receptors. It stimulates resting lymphocytes viainteraction with T11 receptors and subsequent induction of interleukin-2(IL-2) receptors.

Sanderson et al., in Proc. Natl. Acad. Sci., Vol. 83, pgs. 437-440(1986), proposed that the name interleukin 4 be given to eosinophildifferentiation factor based on evidence that it is apparently the sameas B cell growth factor II.

From the foregoing it is evident that the discovery and development ofnew lymphokines could contribute to the development of therapies for awide range of degenerative conditions which directly or indirectlyinvolve the immune system and/or hematopoietic cells. In particular, thediscovery and development of lymphokines which enhance or potentiate thebeneficial activities of known lymphokines would be highly advantageous.For example, the dose-limiting toxicity of IL-2 in tumor therapy couldbe reduced by the availability of a lymphokine or cofactor withpotentiating effects; or, the efficacy of bone marrow transplants couldbe increased by the availability of factors which potentiate theactivities of the colony stimulating factors.

SUMMARY OF THE INVENTION

The present invention is directed to mammalian interleukin-4 (IL-4). Itincludes nucleic acids coding for polypeptides exhibiting IL-4 activity,as well as the polypeptides themselves and methods for their production.The nucleic acids of the invention are defined (1) by their homology tocloned complementary DNA (cDNA) sequences disclosed herein, and (2) byfunctional assays for IL-4 activity applied to the polypeptides encodedby the nucleic acids. As used herein, the term "IL-4 activity" inreference to a protein or a polypeptide means that the protein orpolypeptide exhibits both B-cell growth factor (BCGF) activity and Tcell growth factor (TCGF) activity. For a given mammal, IL-4 activity isdetermined by species specific TCGF and BCGF assays. As explained morefully below, specific embodiments of IL-4 can be further characterizedby additional assays. For example, some forms of murine IL-4 exhibitmast cell growth factor (MCGF) activity; some forms of both human andmurine IL-4 potentiate the TCGF activity of IL-2; some forms of bothmurine and human IL-4 potentiate GM-CSF stimulated proliferation incertain cell types; some forms of both human and murine IL-4 can induceFc-epsilon receptor expression on B cells; and some forms of both humanand murine IL-4 can induce the expression of major histocompatibilitycomplex (MHC) antigens on B cells: The class II DR antigen on human Bcells, and the Ia antigen on mouse B cells.

The invention is based in part on the discovery and cloning of cDNAswhich are capable of expressing proteins having IL-4 activity. cDNAclones of the invention include human cDNA inserts of plasmid vectors"clone 46" (also referred to herein as pcD-2F1-13 or pcD-46) and "clone25" (also referred to herein as pcD-125); and mouse cDNA insert ofplasmid vector pcD-2A-E3. The three vectors are deposited with theAmerican Type Culture Collection (ATCC), Rockville, MD, under ATCCaccession numbers 53337, 67029, and 53330, respectively.

The invention includes nucleic acids having nucleotide sequences whichare effectively homologous to the cDNA clones of the invention and whichexpress IL-4 activity. Nucleic acids and proteins of the invention canbe derived from the above mentioned cDNAs by standard techniques formutating nucleic acid sequences. They can be prepared de novo fromimmune system-derived cell lines, such as T cell hybridomas, whichcontain or can be induced to contain messenger RNA (mRNA) sequencescoding for IL-4. And they can be obtained by probing DNA or RNA extractsor libraries with probes derived from the cDNA clones of the invention.

The term "effectively homologous" as used herein means that thenucleotide sequence is capable of being detected by a hybridizationprobe derived from a cDNA clone of the invention. The exact numericalmeasure of homology necessary to detect nucleic acids coding for IL-4activity depends on several factors including (1) the homology of theprobe to non-IL-4 coding sequences associated with the target nucleicacids, (2) the stringency of the hybridization conditions, (3) whethersingle stranded or double stranded probes are employed, (4) whether RNAor DNA probes are employed, (5) the measures taken to reduce nonspecificbinding of the probe, (6) the nature of the label used to detect theprobe, (7) the fraction of guanidine and cytosine bases in the probe,(8) the distribution of mismatches between probe and target, (9) thesize of the probe, and the like.

Preferably, an effectively homologous probe derived from the cDNA of theinvention is at least fifty percent (50%) homologous to the sequence tobe isolated. More preferably, the effectively homologous probe is atleast seventy-five to eighty percent (75-80%) homologous to the sequenceto be isolated. And most preferably, the effectively homologous probe isat least ninety percent (90%) homologous to the sequence to be isolated.

Homology as the term is used herein is a measure of similarity betweentwo nucleotide (or amino acid) sequences. Homology is expressed as thefraction or percentage of matching bases (or amino acids) after twosequences (possibly of unequal length) have been aligned. The termalignment is used in the sense defined by Sankoff and Kruskal in chapterone of Time Warps, String Edits, and Macromolecules: The Theory andPractice of Sequence Comparison (Addison-Wesley, Reading, MA, 1983).Roughly, two sequences are aligned by maximizing the number of matchingbases (or amino acids) between the two sequences with the insertion of aminimal number of "blank" or "null" bases into either sequence to bringabout the maximum overlap. Given two sequences, algorithms are availablefor computing their homology, e.g. Needleham and Wunsch, J. Mol. Biol.,Vol. 48, pgs. 443-453 (1970); and Sankoff and Kruskal (cited above) pgs.23-29. Also, commercial services are available for performing suchcomparisons, e.g. Intelligenetics, Inc. (Palo Alto, CA).

A preferred embodiment of the invention is the set of glycosylated orunglycosylated human IL-4 proteins and muteins defined by the followingformula:

    ______________________________________                                        Formula I                                                                     ______________________________________                                        X(His)--X(Lys)--X(Cys)--X(Asp)--X(Ile)--X(Thr)--                              X(Leu)--X(Gln)--X(Glu)--X(Ile)--X(Ile)--X(Lys)--                              X(Thr)--X(Leu)--X(Asn)--X(Ser)--X(Leu)--X(Thr)--                              X(Glu)--X(Gln)--X(Lys)--X(Thr)--X(Leu)--X(Cys)--                              X(Thr)--X(Glu)--X(Leu)--X(Thr)--X(Val)--X(Thr)--                              X(Asp)--X(Ile)--X(Phe)--X(Ala)--X(Ala)--X(Ser)--                              X(Lys)--X(Asn)--X(Thr)--X(Thr)--X(Glu)--X(Lys)--                              X(Glu)--X(Thr)--X(Phe)--X(Cys)--X(Arg)--X(Ala)--                              X(Ala)--X(Thr)--X(Val)--X(Leu)--X(Arg)--X(Gln)--                              X(Phe)--X(Tyr)--X(Ser)--X(His)--X(His)--X(Glu)--                              X(Lys)--X(Asp)--X(Thr)--X(Arg)--X(Cys)--X(Leu)--                              X(Gly)--X(Ala)--X(Thr)--X(Ala)--X(Gln)--X(Gln)--                              X(Phe)--X(His)--X(Arg)--X(His)--X(Lys)--X(Gln)--                              X(Leu)--X(Ile)--X(Arg)--X(Phe)--X(Leu)--X(Lys)--                              X(Arg)--X(Leu)--X(Asp)--X(Arg)--X(Asn)--X(Leu)--                              X(Trp)--X(Gly)--X(Leu)--X(Ala)--X(Gly)--X(Leu)--                              X(Asn)--X(Ser)--X(Cys)--X(Pro)--X(Val)--X(Lys)--                              X(Glu)--X(Ala)--X(Asn)--X(Gln)--X(Ser)--X(Thr)--                              X(Leu)--X(Glu)--X(Asn)--X(Phe)--X(Leu)--X(Glu)--                              X(Arg)--X(Leu)--X(Lys)--X(Thr)--X(Ile)--X(Met)--                              X(Arg)--X(Glu)--X(Lys)--X(Tyr)--X(Ser)--X(Lys)--                              X(Cys)--X(Ser)--X(Ser)                                                        ______________________________________                                    

wherein the term X(Xaa) represents the group of synonymous amino acidsto the amino acid Xaa. Synonymous amino acids within a group havesufficiently similar physicochemical properties that substitutionbetween members of the group will preserve the biological function ofthe molecule, Grantham, Science, vol. 185, pgs. 862-864 (1974). It isclear that insertions and deletions of amino acids may also be made inthe above-defined sequence without altering biological function,particularly if the insertions or deletions only involve a few aminoacids, e.g. under ten, and do not remove or displace amino acids whichare critical to a functional conformation, e.g. cysteine residues,Anfinsen, "Principles That Govern The Folding of Protein Chains",Science, Vol. 181, pgs. 223-230 (1973). Proteins and muteins produced bysuch deletions and/or insertions come within the purview of the presentinvention. Whenever amino acid residues of the protein of Formula I arereferred to herein by number, such number or numbers are in reference tothe N-terminus of the protein.

Preferably the synonymous amino acid groups are those defined in TableI. More preferably, the synonymous amino acid groups are those definedin Table II; and most preferably the synonymous amino acid groups arethose defined in Table III.

                  TABLE I                                                         ______________________________________                                        Preferred Groups of Synonymous Amino Acids                                    Amino Acid    Synonymous Group                                                ______________________________________                                        Ser           Ser, Thr, Gly, Asn                                              Arg           Arg, Gln, Lys, Glu, His                                         Leu           Ile, Phe, Tyr, Met, Val, Leu                                    Pro           Gly, Ala, Thr, Pro                                              Thr           Pro, Ser, Ala, Gly, His, Gln, Thr                               Ala           Gly, Thr, Pro, Ala                                              Val           Met, Tyr, Phe, Ile, Leu, Val                                    Gly           Gly, Ala, Thr, Pro, Ser                                         Ile           Met, Tyr, Phe, Val, Leu, Ile                                    Phe           Trp, Met, Tyr, Ile, Val, Leu, Phe                               Tyr           Trp, Met, Phe, Ile, Val, Leu, Tyr                               Cys           Cys, Ser, Thr                                                   His           His, Glu, Lys, Gln, Thr, Arg                                    Gln           Gln, Glu, Lys, Asn, His, Thr, Arg                               Asn           Asn, Gln, Asp, Ser                                              Lys           Lys, Glu, Gln, His, Arg                                         Asp           Asp, Glu, Asn                                                   Glu           Glu, Asp, Lys, Asn, Gln, His, Arg                               Met           Met, Phe, Ile, Val, Leu                                         Trp           Trp                                                             ______________________________________                                    

                  TABLE II                                                        ______________________________________                                        More Preferred Groups of Synonymous Amino Acids                               Amino Acid       Synonymous Group                                             ______________________________________                                        Ser              Ser                                                          Arg              His, Lys, Arg                                                Leu              Leu, Ile, Phe, Met                                           Pro              Ala, Pro                                                     Thr              Thr                                                          Ala              Pro, Ala                                                     Val              Val, Met, Ile                                                Gly              Gly                                                          Ile              Ile, Met, Phe, Val, Leu                                      Phe              Met, Tyr, Ile, Leu, Phe                                      Tyr              Phe, Tyr                                                     Cys              Cys, Ser                                                     His              His, Gln, Arg                                                Gln              Glu, Gln, His                                                Asn              Asp, Asn                                                     Lys              Lys, Arg                                                     Asp              Asp, Asn                                                     Glu              Glu, Gln                                                     Met              Met, Phe, Ile, Val, Leu                                      Trp              Trp                                                          ______________________________________                                    

                  TABLE III                                                       ______________________________________                                        Most Preferred Groups of Synonymous Amino Acids                               Amino Acid        Synonymous Group                                            ______________________________________                                        Ser               Ser                                                         Arg               Arg                                                         Leu               Leu, Ile, Met                                               Pro               Pro                                                         Thr               Thr                                                         Ala               Ala                                                         Val               Val                                                         Gly               Gly                                                         Ile               Ile, Met, Leu                                               Phe               Phe                                                         Tyr               Tyr                                                         Cys               Cys, Ser                                                    His               His                                                         Gln               Gln                                                         Asn               Asn                                                         Lys               Lys                                                         Asp               Asp                                                         Glu               Glu                                                         Met               Met, Ile, Leu                                               Trp               Met                                                         ______________________________________                                    

The invention includes the polypeptides of Formula I with amino acidsubstitutions (between an amino acid of the native human IL-4 and asynonymous amino acid) at a single position or at multiple positions.The term "N-fold substituted" is used to describe a subset ofpolypeptides defined by Formula I wherein the native amino acids havebeen substituted by synonymous amino acids at no more than N positions.Thus, for example, the group of 1-fold substituted polypeptides ofFormula I consists of 559 polypeptides for the preferred groups ofsynonymous amino acids, 189 for the more preferred groups of synonymousamino acids, and 56 for the most preferred groups of synonymous aminoacids. These numbers were arrived at by summing the number of aminoacids of each kind in the native chain times one less than the size ofthe synonymous amino acid group for that amino acid. Preferably thegroup of human IL-4 polypeptides consists of the 10-fold substitutedpolypeptides of Formula I; more preferably they consist of 3-foldsubstituted polypeptides of Formula I; and most preferably they consistof 1-fold substituted polypeptides of Formula I, which in particularincludes the native human IL-4 polypeptide whose sequence is illustratedin Fig. 1C.

As a further example, consider a 1-fold substituted peptide having thesequence:

    Ser-X(Lys)-Cys-X(Ala)

Preferably X(Lys) is the group consisting of Lys and Arg; and mostpreferably, it is the group consisting solely of Lys. Likewise, X(Ala)preferably is the group consisting of Ala and Pro; and most preferably,it is the group consisting solely of Ala. The term "1-fold substituted"in reference to the above sequence defines two groups of peptides, onewith respect to the preferred groups for X(Lys) and X(Ala), and one withrespect to the most preferred groups for X(Lys) and X(Ala). The "1" inthe term "1-fold substituted" means that the peptides of the groupsdiffer from the sequence,

    Ser-Lys-Cys-Ala

by no more than 1 amino acid substitution. The following list is thegroup of 1-fold substituted peptides of the above sequence, with respectto the preferred amino acid groups for X(Lys) and X(Ala):

    Ser-Lys-Cys-Ala

    Ser-Arg-Cys-Ala

    Ser-Lys-Cys-Pro

The sequence Ser-Arg-Cys-Pro is not included because it has 2substitutions. Since the most preferred groups of amino acids for X(Lys)and X(Ala) each only consist of a single amino acid, the group of 1-foldsubstituted peptides of the above sequence with respect to the mostpreferred amino acid group consists solely of the sequence,Ser-Lys-Cys-Ala.

Likewise, the term "N-fold inserted" in reference to the polypeptides ofFormula I is used to describe a set of polypeptides wherein from 1 to Namino acids have been inserted into the sequence defined by Formula I.Preferably, the inserted amino acids are selected from the preferredgroups of synonymous amino acids (Table I) of the amino acids flankingthe insertion; more preferably they are selected from the more preferredgroups of synonymous amino acids (Table II) of the amino acids flankingthe insertion, and most preferably they are selected from the mostpreferred groups of synonymous amino acids (Table III) of the aminoacids flanking the insertion. Thus, for example, one subgroup of thegroup of 1-fold inserted peptides comprises an amino acid insertedbetween the N-terminal X(His) and the adjacent X(Gly). The insertionsdefining the members of this subgroup are preferably selected from thegroup consisting of Pro, Ala, Gly, Thr, Ser, Gln, Glu, Arg, His, andLys; more preferably they are selected from the group consisting of Gly,His, Gln and Arg, and most preferably they are selected from the groupconsisting of His and Gly. Insertions can be made between any adjacentamino acids of Formula I. Since there are 128 possible insertionlocations, and since multiple insertions can be made at the samelocation, a 2-fold inserted polypeptide of Formula I gives rise to16,384 subgroups of polypeptides, and the size of each subgroup dependson the sizes of the synonymous amino acid groups of the amino acidsflanking the insertions.

The term "N-fold deleted" in reference to the polypeptides of Formula Iis used to describe a set of peptides having from 1 to N amino acidsdeleted from the sequence defined by Formula I. Thus, the set of 1-folddeleted polypeptides of Formula I consists of 129 subgroups ofpolypeptides each 128 amino acids in length (128-mers). Each of thesubgroups in turn consists of all the 128-mers defined by the preferred,more preferred, and most preferred synonymous amino acid groups.

The above preferred embodiment of the invention further includesnucleotide sequences effectively homologous to or capable of encodingthe polypeptides of Formula I for the preferred, more preferred, andmost preferred groups of synonymous amino acids. More preferably saidnucleotide sequences are capable of encoding the polypeptides of FormulaI for the preferred, more preferred, and most preferred groups ofsynonymous amino acids.

In particular, the invention includes native human IL-4, the amino acidsequence of which is illustrated in FIG. 1C and all nucleotide sequencescapable of encoding it.

Throughout, standard abbreviations are used to designate amino acids,nucleotides, restriction endonucleases, and the like, e.g. Cohn,"Nomenclature and Symbolism of α-Amino Acids, "Methods in Enzymology,Vol. 106, pgs. 3-17 (1984); Wood et al. Biochemistry: A ProblemsApproach, 2nd ed. (Benjamin, Menlo Park, 1981); and Roberts, "Directoryof Restriction Endonucleases", Methods in Enzymology, Vol. 68, pgs.27-40 (1979).

The present invention is addressed to problems associated with theapplication of immunoregulatory agents to treat medical and/orveterinary disorders. In particular, it provides compounds which alonehave beneficial effects, or which can act in concert with otherlymphokines and immune system mediators to produce beneficial effects.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1A illustrates the nucleotide sequence and deduced amino acidsequence of the insert of vector pcD-2A-E3, which expresses murine IL-4.

FIG. 1B illustrates the nucleotide sequence and deduced amino acidsequence of the insert of vector pcD-125, which expresses human IL-4.

FIG. 1C illustrates the amino acid sequence of purified native humanIL-4 expressed and secreted by COS 7 monkey cells transfected withpcD-125.

FIG. 2A is a map of vector pcD-2A-E3, the insert of which codes murineIL-4.

FIG. 2B is a restriction endonuclease cleavage map of the insert ofvector pcD-2A-E3.

FIG. 2C is a map of vector pcD-46, the insert of which codes human IL-4.

FIG. 2D is a restriction endonuclease cleavage map of the insert ofvector pcD-46.

FIG. 3A illustrates relative TCGF activities (over a range of dilutions)of various culture supernatants including one (curve 1) from pcD-2A-E3transfected COS 7 cells.

FIG. 3B illustrates relative MCGF activities (over a range of dilutions)of various culture supernatants including one (curve 1) from pcD-2A-E3transfected COS 7 cells.

FIG. 3C illustrates the relative degrees of Ia induction produced by theindicated amounts of supernatant from pcD-2A-E3 transfected COS 7 cells(curve 1), C1.Ly1⁺ 2⁻ /9 cells (curve 2), and mock transfected COS 7cells (curve 3).

FIG. 3D graphically illustrates the extent of IgE and IgG₁ induction bysupernatants from pcD-2A-E3 transfected COS 7 cells and various controlsin T cell-depleted mouse spleen cells.

FIG. 4A illustrates the TCGF activities of several pcD-125 transfectionsupernatants and controls as measured by a colorimetric proliferationassay on the factor-dependent human helper T cell line, JL-EBV.

FIG. 4B illustrates the TCGF activities of a pcD-125 transfectionsupernatant and controls as measure by a colorimetric proliferationassay on PHA-stimulated peripheral blood lymphocytes.

FIG. 4C illustrates the TCGF activities of a pcD-125 transfectionsupernatant and controls as measured by tritiated thymidineincorporation by PHA-stimulated peripheral blood lymphocytes.

FIG. 5A is a histogram of cell frequency versus fluorescence intensityfor a control population of stimulated human tonsilar B cells whoseFc-epsilon receptors have been fluorescently labeled.

FIG. 5B is a histogram of cell frequency versus fluorescence intensityfor a population of stimulated human tonsilar B cells which had beenexposed to medium consisting of 0.1% supernatant from pcD-125transfected COS 7 cells and whose Fc-epsilon receptors have beenfluorescently labeled.

FIG. 5C is a histogram of cell frequency versus fluorescence intensityfor a population of stimulated human tonsilar B cells which had beenexposed to medium consisting of 1% supernatant from pcD-125 transfectedCOS 7 cells and whose Fc-epsilon receptors have been fluorescentlylabeled.

FIG. 5D is a histogram of cell frequency versus fluorescence intensityfor a population of stimulated human tonsilar B cells which had beenexposed to medium consisting of 10% supernatant from pcD-125 transfectedCOS 7 cells and whose Fc-epsilon receptors have been fluorescentlylabeled.

FIG. 6A illustrates the nucleotide sequence of a synthetic human IL-4gene useful for expressing native or mutant IL-4s in E. coli.

FIG. 6B is a restriction endonuclease cleavage map of a synthetic humanIL-4 gene inserted in plasmid pUC18.

FIGS. 7A-7F illustrate the double stranded DNA fragments 1A/B through6A/B used to construct a synthetic human IL-4 gene.

FIG. 8 illustrates nucleotide sequences adjacent to the initiator ATGcodon in the E. coli expression vector TAC-RBS. The sequences commenceat an EcoRI restriction site and end with a HindIII site. The ribosomebinding sequence (RBS) showing complementarity to the 3' end of 16Sribosomal RNA is underlined, and the ATG initiator codon is underlined.

FIG. 9 illustrates histograms of cell frequency versus fluorescenceintensity for populations of cells derived from a patient with barelymphocyte syndrome. The cells were stained with fluorescently labeledanti-DR monoclonal antibodies.

FIG. 10 illustrates the 215 nm absorption profile in the final humanIL-4 purification step, which consisted of reversed-phase HPLC on a C-4column.

FIG. 11 is a construction map of plasmid pEBV-178 containing human IL-4cDNA.

FIG. 12 is a construction map of plasmid TRPC11.

FIG. 13A is a construction map of plasmid pMF-alpha8.

FIG. 13B illustrates the TCGF activities of several transfectionsupernatants from yeast cultures expressing native human IL-4 andvarious muteins thereof.

DETAILED DESCRIPTION OF THE INVENTION

The present invention includes glycosylated or unglycosylated mammalianpolypeptides which exhibit IL-4 activity, and which are derivable fromthe IL-4 polypeptides disclosed herein using standard proteinengineering techniques. The invention also includes nucleic acids havingsequences capable of coding for the polypeptides of the invention, andnucleic acids whose sequences are effectively homologous to the cDNAclones of the invention. Finally, the invention includes methods ofmaking the glycosylated or unglycosylated polypeptides of the inventionwhich utilize the nucleotides sequences disclosed herein, and methods ofusing the polypeptides of the invention.

Below techniques for making, using, and identifying the polypeptides andnucleic acids of the invention are discussed in general terms.Afterwards several specific examples are provided wherein the generaltechniques are applied using specific cell types, vectors, reagents, andthe like.

I. De Novo Preparation of IL-4 cDNA

A variety of methods are now available for de novo preparation andcloning of cDNAs, and for the construction of cDNA libraries, e.g.recent reviews are given by Doherty, "Cloning and Expression of cDNA",Chapter 10 in Gottesman, Ed. Molecular Cell Genetics (John Wiley & Sons,New York, 1985); and Brandis et al., "Preparation of cDNA Libraries andthe Detection of Specific Gene Sequences", in Setlow et al., Eds.Genetic Engineering, Vol. 8, pgs. 299-316 (Plenum Press, New York,1986).

By way of example, total mRNA is extracted (e.g., as reported by Berger,S. et al., Biochemistry 18 5143-5149 [1979]) from cells (e.g., anontransformed human T-cell source) producing polypeptides exhibitingthe desired activity. The double-stranded cDNAs from this total mRNA canbe constructed by using primer-initiated reverse transcription (Verme,I., Biochem. Biophys. Acta, Vol. 473, pgs. 1-38 [1977]) to make firstthe complement of each mRNA sequence, and then by priming for secondstrand synthesis (Land, H. et al., Nucleic Acids Res., 9: 2251-2266[1981]). Subsequently, the cDNAs can be cloned by joining them tosuitable plasmid or bacteriophage vectors (Rougeon, F. et al., NucleicAcids Res., 2, 2365-2378 [1975]) or Scherer, G. et al., Dev. Biol. 86,438-447 [1981]) through complementary homopolymeric tails (Efstratiadis,A. et al., Cell, 10, 571-585 [1977]) or cohesive ends created withlinker segments containing appropriate restriction sites (Maniatis etal., Molecular Cloning: A Laboratory Manual, Cold Spring HarborLaboratory, N.Y. 1982), and then transforming a suitable host. (Seegenerally, Efstratiadis, A., and Villa-Kormaroff, L., "Cloning of doublestranded cDNA" in Setlow, J. and Hollaender, A. (eds.) GeneticEngineering, Vol, 1, Plenum Publishing Corp., N.Y., U.S.A. [1982].)

A preferred source of mRNA encoding the desired polypeptides are cellswhose supernatants contain the B-cell, T-cell and/or mast cellstimulating activities, or other activities associated with thepolypeptides of the present invention. One such line is the mouse T-cellline Cl.Lyl⁺ 2⁻ /9 (A.T.C.C. Accession No. CRL8179) (Nabel, G. et al.,Nature 291:332-334 (1981)). In general, suitable T-cells can be obtainedfrom a variety of sources, such as mammalian (e.g. human) spleen,tonsils and peripheral blood. T-cell clones, such as those isolated fromperipheral blood T-lymphocytes, may also be used (see, ResearchMonographs in Immunology, eds. von Doehmer, H. and Haaf, V.; Section D:"Human T-Cell Clones", vol.8, pgs. 243-333; Elsevier Science Publishers,N.Y. [1985]).

Production of mRNAs capable of coding for IL-4 by such cells can beconfirmed by microinjection of the extracted mRNA into oocytes ofXenopus laevis. This microinjection technique is described more fullybelow, and is disclosed generally in Colman et al., "Export of Proteinsfrom Oocytes of Xenopus Laevis", Cell, Vol. 17, pgs. 517-526 (1979); andManiatis et al. Molecular Cloning: A Laboratory Manual, pgs. 350-352(Cold Spring Harbor Laboratory, New York, 1982).

If the mRNAs coding for a desired IL-4 make up a very small fraction ofthe total mRNA steps may be necessary to enrich the fractionalconcentration in order to make the screening procedure for detectingcDNA clones of interest practical. Such procedures are standard in theart and are disclosed in the examples below and in several papers andreferences, such as Maniatis et al., pgs. 225-228, cited above; Suggs etal., Proc Natl. Acad. Sci., Vol. 78, pgs. 6613-6617 (1981); Davis etal., Proc. Natl. Acad. Sci., Vol. 81, pgs. 2194-2198 (1984) or the like.

A preferred method of de novo preparation of IL-4 cDNAs relies onfunctional expression of the cDNAs in pcD expression system developed byOkayama and Berg, disclosed in Mol. Cell. Biol., Vol. 2, pgs. 161-170(1982); and Vol. 3. pgs. 280-289 (1983), and available from Pharmacia(Piscataway, N.J.). Accordingly, these references are incorporated byreference. The pcD expression vector contains the SV40 early promoter,late splicing junction, and the replication origin. This vector permitsexpression of cDNA inserts in COS 7 monkey cells which provide T antigenfor replication of the pcD plasmid. Screening of cDNA libraries includestransfection of pools of plasmid DNA into COS 7 cells usingDEAE-Dextran. Since lymphokines, and in particular IL-4s, are secretedproteins, the supernatants from the transfected cells can be assayed forbiological activity after incubation for several days. Positive poolsare further divided to identify single cDNA clones which give biologicalactivity after transfection.

Briefly, the Okayama and Berg expression vector is constructed asfollows. Polyadenylated mRNA is annealed to a polydeoxythymidylic acid(oligo dT) tail attached to the protruding strand of a KpnI digestedpBR322 plasmid containing the SV40 early promoter region. That is, theentire vector serves as a primer for cDNA synthesis. After cDNAsynthesis, 3' polydeoxycytidylate (oligo dC) tails are attached, followby Hind III digestion, which lops off (at a unique Hind III site) afragment of the SV40 DNA to which one of the oligo dC tails is attached.The SV40 early promoter remains intact, and fortuitously occurring HindIII sites of the insert are affected minimally because the hybridcDNA/RNA is resistant to Hind III digestion. A separately constructedHind III fragment having a 3' polyguanidylated (oligo dG) tail isannealed to the sticky end left by the Hind III digestion. The vector iscircularized and treated with E. coli RNase H, DNA polymerase I, and DNAligase to replace the RNA strand with DNA. The vectors are cloned in E.coli to form the cDNA library. The SV40 elements permit the vectors tobe expressed in eucaryotic cells as well as procaryotic cells, andparticularly in mammalian cells, such as COS7 monkey cells or Chinesehamster ovary (CHO) cells.

Once the cDNA library in the Okayama/Berg plasmid vector has beencompleted, the cDNA clones are collected, and random pools checked forthe presence of the desired cDNAs by standard procedures, e.g. hybridselection, detection of antigenic determinants on expressed products,and/or functional assays. Positive pools can then be probed with a cDNAfrom an induced T cell line. Thereafter, the positive, probed pools aredivided into individual clones which are further tested by transfectioninto a suitable host (such as a mammalian cell culture), and the hostsupernatant assayed for activity.

II. Preparation of IL-4 cDNAs Via Hybridization Probes Derived fromDisclosed cDNAs

The cDNAs disclosed herein can be used as probes to identify homologoussequences in different cell types, as an alternative to de novoisolation and cloning of the IL-4 coding nucleotides. Standardtechniques are employed, e.g. Beltz et al., "Isolation of MultigeneFamilies and Determination of Homologies by Filter HybridizationMethods," Methods in Enzymology, Vol. 100, pgs. 266-285. (1983); andCallahan et al., "Detection and Cloning of Human DNA Sequences Relatedto the Mouse Mammary Tumor Virus Genome," Proc. Natl. Acad. Sci., Vol.79, pgs. 5503-5507 (1982), the former reference being incorporated byreference. Basically, the cDNAs of the invention are used to constructprobes (using standard techniques, e.g. see Maniatis et al., citedabove) for screening at low hybridization stringencies genomic or cDNAlibraries (again, constructed by standard techniques) of a cell typesuspected of producing IL-4. Standard screening procedures are employed,e.g. Grunstein et al., Proc. Natl. Acad. Sci., Vol. 72, pgs. 3961-3965(1975); or Benton et al., Science, Vol. 196, pgs. 180-183 (1977).

As described more fully below, human IL-4 was isolated by a murine IL-4probe. Subsequent analysis indicated about 70% homology between selectedregions of the human and mouse cDNAs. Given the evolutionary distancebetween mice and humans it is believed that most, if not all, mammalianIL-4 genes are detectable by probes constructed from one or more cDNAsof the invention, Wilson et. al. "Biochemical Evolution", Ann. Rev.Biochem., Vol. 46, pgs. 573-639 (1977); Kimura, "The Neutral Theory ofMolecular Evolution," Chapter 11 in Nei and Koehn, Eds. Evolution ofGenes and Proteins (Sinauer Associates, Sunderland, MA, 1983).

III. Preparation of Mutant IL-4s by Protein Engineering

Once nucleic acid sequence and/or amino acid sequence information isavailable for a native protein a variety of techniques become availablefor producing virtually any mutation in the native sequence. Shortle, inScience, Vol. 229, pgs. 1193-1201 (1985), review techniques for mutatingnucleic acids which are applicable to the present invention. Preferably,mutants of the native IL-4s, i.e. IL-4 muteins, are produced bysite-specific oligonucleotide-directed mutagenesis, e.g. Zoller andSmith, Methods in Enzymology, Vol. 100, pgs. 468-500 (1983); Mark etal., U.S. Pat. No. 4,518,584 entitled "Human Recombinant Interleukin-2Muteins," which are incorporated by reference; or by so-called"cassette" mutagenesis described by Wells et al., in Gene, Vol. 34, pgs.315-323 (1985); and Estell et al., Science, Vol. 233, pgs. 659-663(1986); and also described essentially by Mullenbach et al., J. Biol.Chem., Vol. 261, pgs. 719-722 (1986), and Feretti et al., Proc. Natl.Acad. Sci., Vol. 83, pgs.. 597-603 (1986). In sections below thenotation used by Estell et al. (cited above) to identify muteins isfollowed and generalized. For example, "human IL-4 mutein Leu⁸² " (orsimply "Leu⁸² " if the native protein is understood from the context)indicates a polypeptide whose amino acid sequence is identical to thatof the native protein except for position 82 with respect to theN-terminus. At that position Leu has been substituted for Phe. Where themutein contains more than one substitution, e.g. Leu for Phe at position82, and Asp for Asn at position 111, the mutein is referred to as humanIL-4 mutein (Leu⁸², Asp¹¹¹). Deletions are indicated by "Δ's". Forexample, a mutein lacking Gln at position 71 is referred to as humanIL-4 mutein Δ⁷¹. Insertions are indicated by "IS(Xaa)'s". For example, amutein with a Leu inserted after Gln at position 71 is referred to ashuman IL-4 mutein IS⁷¹ (Leu). Thus, human IL-4 mutein (Ser¹³, Δ⁷¹, IS⁹⁴(Gly)) represents the native human IL-4 sequence which has been modifiedby replacing Thr by Ser at position 13, deleting Gln at position 71, andinserting Gly immediately after Ala at position 94. Insertion ofmultiple amino acids at the same site is indicated by IS^(i) (Xaa₁ -Xaa₂-Xaa₃ -. . . ) , where Xaa₁ -Xaa₂ -Xaa₃ . . . is the sequence insertedafter position i. N-terminal additions are indicated by superscript "O",e.g. IS⁰ (Xaa), and a sequence of deletions, for example of amino acids6-10, is designated either as Δ⁶⁻¹⁰ or as (Δ⁶, Δ⁷, Δ⁸, Δ⁹, Δ¹⁰).

Most preferably cassette mutagenesis is employed to generate human IL-4muteins. As described more fully below, a synthetic human IL-4 gene hasbeen constructed with a sequence of unique (when inserted in aappropriate vector) restriction endonuclease sites spaced approximatelyuniformly along the gene. The unique restriction sites allow segments ofthe gene to be conveniently excised and replaced with syntheticoligonucleotides (i.e. "cassettes") which code for desired muteins.

Determination of the number and distribution of unique restriction sitesentails the consideration of several factors including (1) preexistingrestriction sites in the vector to be employed in expression, (2)whether species or genera-specific codon usage is desired, and (3) theconvenience and reliability of synthesizing and/or sequencing thesegments between the unique restriction sites.

IV. Biological Properties and Assays for IL-4 Activity.

Mammalian IL-4 of the invention is defined in terms of biologicalactivities and/or homology with the disclosed embodiments. MammalianIL-4s of the invention include proteins and muteins (of the disclosednative polypeptides) which are homologous to the disclosed nativepolypeptides and which exhibit both BCGF activity and TCGF activity.Mammalian IL-4s of the invention are alternatively defined by theirbiological activities (defined more fully below) which include BCGFactivity and TCGF activity (which is collectively referred to herein asIL-4 activity) as well as at least one or more activities selected fromthe group of activities consisting of MHC antigen induction activity,Fc-epsilon receptor induction activity, GM-CSF stimulated granulocytecolony growth potentiating activity, interleukin-2 TCGF potentiatingactivity, and IgG₁ and IgE induction activity.

It is believed that IL-4s are species specific in their activities. Thatis, for example, human IL-4 exhibits TCGF activity as assayed by human Tcell lines, but not as assayed by murine T cell lines. And conversely,murine IL-4 exhibits TCGF activity as assayed by murine T cell lines,but not as assayed by human T cell lines, Mosmann et al.,"Species-Specificity of T Cell Stimulating Activities of IL-2 and BSF-1(IL-4): Comparison of Normal and Recombinant, Mouse and Human IL-2 andBSF-1 (IL-4), J. Immunol., Vol. 138, pgs. 1813-1816 (1987)".

A. TCGF Activity

Several standard assays have been described for TCGF activity, e.g.Devos et al., Nucleic Acids Research, Vol. 11, pgs. 4307-4323 (1983);Thurman et al., J. Biol. Response Modifiers, Vol. 5, pgs 85-107 (1986);and RobertGuroff et al., Chapter 9 in Guroff, Ed. Growth and MaturationFactors (John Wiley, New York, 1984). Accordingly these references areincorporated by reference for their descriptions of TCGF activityassays. Generally, the TCGF assays are based on the ability of a factorto promote the proliferation of peripheral T lymphocytes or IL-2dependent T cell lines, e.g. Gillis et al. J. Immunol., Vol. 120, pg.2027 (1978). Proliferation can be determined by standard techniques,e.g. tritiated thymidine incorporation, or by colorimetric methods,Mosmann, J. Immunol. Meth., Vol. 65, pgs. 55-63 (1983).

By way of example, human TCGF activity can be assayed by the followingsteps: (1) washed human peripheral blood lymphocytes (about 2×10⁵ in 50microliters) previously stimulated with phytohemagglutinin (PHA) for 7days and subsequently cultured for 7 days with IL-2 are added to amicrotiter well; (2) dilutions (50 microliter) of the TCGF-containingmaterial are added to each well; (3) the lymphocytes are incubated 72hours at 37° C.; (4) tritiated thymidine (about 20 microliters, 20microcuries/ml) is added to each well; and (5) cells are harvested ontofilter paper strips, washed, and counted in a scintillation counter.

As described more fully in the examples, some forms of IL-4 have thecapability of potentiating the TCGF activity of IL-2. "Potentiation" asused herein in reference to such activity means that the maximal levelof proliferation in a TCGF assay system caused by IL-2 is increased bythe addition of IL-4.

B. BCGF Activity

BCGF activity is defined by an assay disclosed by Howard et al., J. Exp.Med, Vol. 155, pgs. 914-923 (1982), which is incorporated herein byreference. Assays for BCGF are reviewed generally by Howard and Paul, inAnn. Rev. Immunol., Vol. 1, pgs. 307-333 (1983). Briefly, BCGF activityis measured by the degree to which purified resting B cells arestimulated to proliferate in the presence of a submitogenicconcentration anti-IgM, or like antigen. By way of example, assay ofhuman BCGF activity can be carried out by the following steps:

Enriched B cell populations are obtained from peripheral blood, spleen,tonsils, or other standard sources by Ficoll/Hypaque density gradientcentrifugation (e.g. Pharmacia) and two cycles of rosetting with2-aminoethylisothiouronium bromide-treated sheep erythrocytes toeliminate T cells. Such B cell preparations should contain greater than95% surface Ig⁺ cells and greater than 95% cells positive for humanB-cell specific antigen, as determined by the anti-human B-cell specificmonoclonal antibody B1 available from Coulter (Hialeah, FL). T cellcontamination should be less than 1% as determined by staining withanti-Leu-1 monoclonal antibodies (Becton-Dickinson, Mountain View, CA)or OKT 11 antibodies (Ortho Diagnostics, Westwood, MA). 3 millilitercultures of such B lymphocytes (about 5×10⁵ per ml in Yssel's medium,Yssel et al., J. Immunol. Meth., Vol. 65, pgs. 55-63 (1984), which isincorporated by reference) are activated by either Staphylococcus aureusCowan I strain (SAC) (e.g. 0.01% solution of SAC, which is availablefrom Calbiochem under the tradename Pansorbin, or which can be preparedas described by Falkoff et al., J. Immunol., Vol. 129, pg. 97-102(1982)) or anti-IgM antibodies (e.g. BRL, Gaithersburg, MD) coupled tobeads, e.g. 5 microgram/ml of Immunobeads available from Bio-Rad(Richmond, CA). The B cells are cultured either for 24 hours (in thecase of SAC) or 72 hours (for anti-IgM beads) and then repurified byFicoll/Hypaque density centrifugation to remove SAC particles, beads,nonviable cells, and the like. B cell proliferation is measured byplating about 5×10⁴ B lymphocytes in 50 microliters of medium in 0.2 mlflat-bottomed microtiter wells. Various dilutions of the materialssuspected of having BCGF activity are added in a final volume of 50microliters. Tritiated thymidine incorporation is determined after 48hours (anti-IgM cultures) or 72 hours (SAC cultures). Similar assays arealso disclosed by Muraguchi et al., J. Immunol., Vol. 129, pgs.1104-1108 (1982); and Yoshizaki et al., J. Immunol., Vol. 128, pgs.1296-1301 (1981).

C. MHC Antigen Induction.

It has been demonstrated that IL-4 can induce the expression of MHCantigens (e.g., Ia in mice) in various cell types of the immune system,particularly B cells, e.g. Zlotnik et al., J. Immunol., Vol. 138, pgs.4275-4279 (1987). Roehm et al., in J. Exp. Med., Vol. 160, pgs. 679-694,presented evidence that a factor exhibiting BCGF activity was alsocapable of inducing the expression of MHC antigens on normal resting Bcells. Assays for MHC antigen induction are generalizations of theassays for murine B cells presented in that reference (accordingly it isincorporated by reference). Briefly, immune system cells are exposed toIL-4, and then expression of particular MHC antigens on the cells'surfaces are determined by labeled antibodies specific for that antigen.The degree of induction is determined by comparison of the induced cellswith controls. Several different antibodies can be employed for anygiven species. Several hybridomas are available from the ATCC whichproduce monoclonal anti-MHC antigen antibodies, and several areavailable commercially (for example, anti-HLA-DR produced by hybridomasunder ATCC accession numbers HB103, HB109, HB110, or HB151;anti-I-A^(b),d produced by hybridoma under ATCC accession number HB35;anti-HLA-DR L243 available from Becton Dickinson (Mountain View, CA); orthe like). Some routine experimentation may be required to adapt theassay to a particular species, and to optimize conditions to give themost sensitive read out of MHC antigen levels. For the human MHC antigeninduction assay, purified B cells can be prepared as described above, orby similar techniques. Alternatively, MHC induction can be assayed onunpurified preparations of spleen cells. Antibody-labeled cells arepreferably detected flow cytometrically, e.g. on a Becton DickinsonFACS-type instrument, or the equivalent.

D. MCGF Activity

It is believed that IL-4s generally exhibit MCGF activity. However,because of the lack of adequate assay techniques MCGF activity has onlybeen demonstrated for rodent IL-4. Murine IL-4 MCGF assays are based onthe proliferation of factor dependent murine mast or basophil celllines. In particular, MCGF activity can be assayed with the murine mastcell line MC/9, which is deposited with the ATCC under accession numberCRL 8306, and is described in U.S. Pat. No. 4,559,310 (which isincorporated by reference) and in Nabel et al., Cell, Vol. 23, pg. 19(1981). Murine MCGF assays are also described by Ihle et al., in J.Immunol., Vol. 127, pg. 794 (1981).

Preferably MCGF activity is determined by the colorimetric assay ofMosmann (cited above) with the use of MC/9 cells. Briefly, MC/9 cellsare cultured in flat-bottom Falcon microtiter trays (10⁴ cells/well) inDulbecco's modified medium supplemented with 4% fetal calf serum 50 μM2-mercaptoethanol, 2 mM glutamine, nonessential amino acids, essentialvitamins, and varied concentrations of test supernatants in a finalvolume of 0.1 ml. Fifty micrograms of3-(4.5-dimethylthiazol-2-yl)-2.5-diphenyl tetrazolium bromide (Sigma) in10 μl of phosphate-buffered saline were added to each cell culture aftera 20-hr incubation. Four hours later, 0.1 ml of 0.04M HCl in isopropanolwas added to solubilize the colored formazan reaction product. Theabsorbance at 570 nm (reference 630 nm) is measured on a DynatekMicroelisa Autoreader (MR580), or similar instrument.

E. Fc-epsilon Receptor Induction.

It has been discovered that IL-4 induces Fc-epsilon expression on Bcells and on T cells, but particularly on human B cells stimulated byanti-IgM antibodies, or like antigen. It has also been discovered thatgamma interferon specifically inhibits IL-4 induced Fc-epsilonexpression on B cells.

Preferably, the assay for Fc-epsilon receptor induction proceedsinitially as for the BCGF assay. That is, purified B cell are obtainedwhich are then stimulated with anti-IgM antibody (or like antigen) andare exposed to IL-4. Finally the cells are assayed for Fc-epsilonreceptors.

Several assays are available for quantifying Fc-epsilon receptors oncell surfaces, e.g. Yodoi and Ishizaka, J. Immunol., Vol. 122, pgs.2577-2583 (1979); Hudak et al., J. Immunol Meth., Vol. 84, pgs. 11-24(1985); and Bonnefoy et al., J. Immunol. Meth., Vol. 88, pgs. 25-32(1986). In particular, Fc-epsilon receptors can be measured flowcytometrically with labeled monoclonal antibodies specific for thereceptors, e.g. using a Becton Dickinson FACS-IV, or like instrument.Fc-epsilon receptor specific monoclonals can be constructed usingconventional techniques.

F. IgG₁ and IgE Induction.

IL-4 induces the secretion of IgE and IgG₁ isotypes inlipopolysaccharide (LPS)-activated B cells, e.g. Coffman et al., J.Immunol., Vol. 136, pgs. 4538-4541 (1986); Sideras, et al., Eur. J.Immunol., Vol. 15, pgs. 586-593 (1985). These activities can be measuredby standard immunoassays for antibody isotype, such as described byCoffman et al., J. Immunol., Vol. 136, pgs. 949-954 (1986). Briefly, Bcells are LPS activated by culturing them with, for example, about 4micrograms/ml of Salmonella typhimurium LPS (available from Sigma) orabout 50 microgram/ml LPS extracted from E. coli 055 (as described bySideras et al., cited above). After 4-8 days culture supernatants areharvested for assaying. Standard isotype-specific ELISA-type assays canbe used to measure the various isotype concentrations. Anti-isotypeantibodies for the assay are available commercially, or can be obtainedfrom the ATCC.

G. Colony Stimulating Factor (CSF) Activity.

To determine CSF activity, hemopoietic cells, e.g. bone marrow cells orfetal cord blood cells, are made into a single cell suspension. Theindividual cells are then "immobilized" in a semi-solid (agar) orviscous (methylcellulose) medium containing nutrients and usually fetalcalf serum. In the presence of an appropriate stimulating factor,individual cells will proliferate and differentiate. Since the initialcells are immobilized, colonies develop as the cells proliferate andmature. These colonies can be scored after 7-14 days, Burgess, A.,Growth Factors and Stem Cells, pgs. 52-55, Academic Press, New York[1984]. (For specific application to the growth of granulocytes andmacrophages, see Bradely, T. and Metcalf, D., Aust. J. Exp. Biol. Med.Sci. Vol. 44, pgs. 287-300 [1966], and see generally Metcalf, D.,Hemopoietic Colonies, Springer-Verlag, Berlin [1977]). If desired,individual colonies can be extracted, placed on microscope slides, fixedand stained with Wright/Geimsa (Todd-Sanford, Clinical Diagnosis byLaboratory Methods, 15th Edition, Eds. Davidson and Henry [1974]).Morphological analysis of cell types present per single colony can thenbe determined.

Bone marrow cells collected from patients with nonhematologic diseaseare layered over Ficoll (type 400, Sigma Chemical Co., St. Louis, MO),centrifuged (2,000 rpm's, 20 min), and the cells at the interfaceremoved. These cells are washed twice in Iscove's Modified Dulbecco'sMedium containing 10% fetal calf serum (FCS), resuspended in the samemedium and the adherent cells removed by adherence to plastic Petridishes. The nonadherent cells are added at 10⁵ cells/ml to Iscove'sMedium containing 20% FCS, 50 μM 2-mercaptoethanol, 0.9% methylcelluloseand varied concentrations of either supernatants known to contain colonystimulating activity or test supernatants. One ml aliquots are plated in35 mm petri dishes and cultured at 37° C. in a fully humidifiedatmosphere of 6% CO₂ in air. Three days after the initiation of theculture, 1 unit of erythropoietin is added to each plate.Granulocyte-macrophage colonies and erythroid bursts are scored at 10-14days using an inverted microscope.

Cord blood cells collected in heparin are spun at 2,000 rpm's for 6 min.The white blood cells at the interface between the plasma and red bloodcell peak are transferred to a tube containing 0.17N ammonium chlorideand 6% FCS. After 5 min on ice, the suspension is underlaid with 4 mlFCS and centrifuged for 6 mins at 2,000 rpm. The cell pellet is washedwith Dulbecco's phosphate buffered saline and put through the Ficoll andplastic adherence steps as described above for bone marrow cells. Thelow density nonadherent cells are collected and placed at 10⁵cells/culture in the semi-solid culture medium as described above.

At the end of the assays, the cellular composition is determined afterapplying the individual colonies to glass slides and staining withWright-Giemsa. Eosinophils are determined by staining with Luxol FastBlue (Johnson, G. and Metcalf, D., Exp. Hematol. Vol. 8, pgs. 549-561[1980]).

"Potentiation" as used herein in reference to GM-CSF stimulatedgranulocyte growth means that granulocyte colonies in the assaysdescribed above are larger when GM-CSF is used with IL-4 than whenGM-CSF is used to stimulate colony growth alone.

V. Purification and Pharmaceutical Compositions

The polypeptides of the present invention expressed in E. coli, in yeastor in other cells can be purified according to standard procedures ofthe art, including ammonium sulfate precipitation, fractionation columnchromatography (e.g., ion exchange, gel filtration, electrophoresis,affinity chromatography, etc.) and ultimately crystallization (seegenerally "Enzyme Purification and Related Techniques", Methods inEnzymology, 22:223-577 [1977] and Scopes, R., Protein Purification:Principles and Practice, Springer-Verlag, New York [1982]). Oncepurified, partially or to homogeneity, the polypeptides of the inventionmay be used for research purposes, e.g., as a supplement to cell growthmedia (e.g., minimum essential medium Eagle, Iscove's modified DulbeccoMedium or RPMI 1640; available from Sigma Chemical Company (St. Louis,MO) and GIBCO Division (Chagrin Falls, OH) and as an antigenic substancefor eliciting specific immunoglobulins useful in immunoassays,immunofluorescent stainings, etc. (See generally, Immunological Methods,Vols. I and II, Eds. Lefkovits, I. and Pernis, B., Academic Press, NewYork, N.Y. [1979 and 1981]; and Handbook of Experimental Immunology, ed.Weir, D., Blackwell Scientific Publications, St. Louis, MO [1978].)

The polypeptides of the present invention may also be used inpharmaceutical compositions, e.g., to enhance natural defense againstvarious infections. Thus, patients with rheumatoid arthritis, in need ofa transplant, or with immunodeficiency caused by cancer chemotherapy,advanced age, immunosuppressive agents, etc., may be treated with suchpolypeptides. The compositions can selectively stimulate variouscomponents of the immune system, either alone or with other agents wellknown to those skilled in the art. In particular, the compositions mayinclude other immune-reactive agents, such as lymphokines (e.g. IL-1,IL-2, etc.), any of the colony stimulating factors, immunoglobulins,etc., in view of the potentiating activities of the polypeptides of thepresent invention. The polypeptides will also find use in situations (invivo or in vitro) in which enhanced cellular proliferation orimmunoglobulin production is desired.

For preparing pharmaceutical compositions containing the polypeptidesdescribed by this invention, these polypeptides are compounded byadmixture with preferably inert, pharmaceutically acceptable carriers.Suitable carriers and processes for their preparation are well known inthe art (see, e.g., Remington's Pharmaceutical Sciences and U.S.Pharmacopeia: National Formulary, Mack Publishing Company, Easton, PA[1984]). The preferred course of administration is parenteral and caninclude use of mechanical delivery systems.

Preferably, the pharmaceutical composition is in unit dosage form. Insuch form, the preparation is subdivided into unit doses containingappropriate quantities of the active component. The quantity of activecompound in a unit dose of preparation may be varied or adjusted from 1μg to 100 mg, according to the particular application and the potency ofthe active ingredient. The composition can, if desired, also containother therapeutic agents.

The dosages may be varied depending upon the requirement of the patient,the severity of the condition being treated and the particular compoundbeing employed. The term "effective amount" as used herein is meant totake these factors into account when dosages are considered.Determination of the proper dosage for a particular situation is withinthe skill of the art. Generally, treatment is initiated with smallerdosages which are less than the optimum dose of the compound.Thereafter, the dosage is increased by small increments until theoptimum effect under the circumstances is reached. For convenience, thetotal daily dosage may be divided and administered in portions duringthe day.

VI. Expression Systems

A wide range of expression systems (i.e. host-vector combinations) canbe used to produce the proteins and muteins of present invention.Possible types of host cells include but are not limited to cells frombacteria, yeast, insects, mammals, and the like. Optimizing theexpression of a particular protein or mutein depends on many factors,including (1) the nature of the protein or mutein to be expressed, e.g.the expressed product may be poisonous to some host systems, (2)whether, and what type of, post-translational modifications are desired,e.g. the extent and kind of glycosylation desired may affect the choiceof host, (3) the nature of the 5' and 3' regions flanking the codingregion of the protein or mutein of interest, e.g. selection of promotersand/or sequences involved in the control of translation is crucial forefficient expression, (4) whether transient or stable expression issought, (5) the ease with which the expressed product can be separatedfrom the proteins and other materials of the host cells and/or culturemedium, (6) the ease and efficiency of transfecting hosts whichtransiently express the protein or mutein of interest, (7) the scale ofcell culture employed to express the protein or mutein of interest, (8)whether the protein or mutein of interest is expressed fused to afragment of protein endogenous to the host, and like factors.

In general prokaryotes are preferred for cloning the DNA sequences ofthe invention. General guides for implementing prokaryotic expressionsystems are provided by Maniatis et al., Molecular Cloning: A LaboratoryManual (Cold Spring Harbor Laboratory, N.Y., 1982); Perbal, A PracticalGuide to Molecular Cloning (John Wiley & Sons, N.Y., 1984); Glover, DNACloning: A Practical Approach, Vol. I and II (IRL Press, Oxford, 1985);and de Boer et al., "Strategies for Optimizing Foreign Gene Expressionin Escherichia coli," in Genes: Structure and Expression, Kroon, ed.(John Wiley & Sons, N.Y. , 1983). For example, E. coli K12 strain 294(ATCC No. 31446) is particularly useful. Other microbial strains whichmay be used include E. coli strains such as E. coli B, and E. coli X1776(ATCC No. 31537). These examples are, of course, intended to beillustrative rather than limiting.

Prokaryotes may also be used for expression. The aforementioned strains,as well as E. coli W3110 (Fs⁻, λ⁻, prototrophic, ATCC No. 27325),bacilli such as Bacillus subtilus, and other enterobacteriaceae such asSalmonella typhimurium or Serratia marcesans, and various pseudomonasspecies may be used.

In general, plasmid vectors containing replicon and control sequenceswhich are derived from species compatible with the host cell are used inconnection with these hosts. The vector ordinarily carries a replicationsite, as well as marking sequences which are capable of providingphenotypic selection in transformed cells. For example, E. coli istypically transformed using pBR322, a plasmid derived from an E. colispecies (Bolivar, et al., Gene Vol. 2, pg. 95 (1977)). pBR322 containsgenes for ampicillin and tetracycline resistance and thus provides easymeans for identifying transformed cells. The pBR322 plasmid, or othermicrobial plasmid must also contain, or be modified to contain,promoters which can be used by the microbial organism for expression ofits own proteins. Those promoters most commonly used in recombinant DNAconstruction include the β-lactamase (penicillinase) and lactosepromoter systems (Chang et al, Nature, Vol. 275, pg. 615 (1978);Itakura, et al, Science, Vol. 198, pg. 1056 (1977); (Goeddel, et alNature Vol. 281, pg. 544 (1979)) and a tryptophan (trp) promoter system(Goeddel, et al, Nucleic Acids Res., Vol. 8, pg. 4057 (1980); EPO ApplPubl No. 0036776). While these are the most commonly used, othermicrobial promoters have been discovered and utilized, and detailsconcerning their nucleotide sequences have been published, enabling askilled worker to ligate them functionally with plasmid vectors(Siebenlist, et al, Cell Vol. 20, pg. 269 (1980)).

In addition to prokaryotes, eukaryotic microbes, such as yeast culturesmay also be used. Saccharomyces cerevisiae, or common baker's yeast isthe most commonly used among eukaryotic microorganisms, although anumber of other strains are commonly available. For expression inSaccharomyces, the plasmid YRp7, for example, (Stinchcomb, et al,Nature, Vol. 282, pg 39 (1979); Kingsman et al, Gene, Vol. 7, pg. 141(1979); Tschemper, et al, Gene, Vol. 10, pg. 157 (1980)) is commonlyused. This plasmid already contains the trp1 gene which provides aselection marker for a mutant strain of yeast lacking the ability togrow in tryptophan, for example ATCC No. 44076 or PEP4-1 (Jones,Genetics, Vol. 85, pg. 12 (1977)). The presence of the trp1 lesion as acharacteristic of the yeast host cell genome then provides an effectiveenvironment for detecting transformation by growth in the absence oftryptophan.

Suitable promoting sequences in yeast vectors include the promoters for3-phosphoglycerate kinase (Hitzeman, et al., J. Biol. Chem., Vol. 255,pg. 2073 (1980)) or other glycolytic enzymes (Hess, et al, J. Adv.Enzyme Reg., Vol. 7, pg. 149 (1968); Holland, et al, Biochemistry, Vol.17, pg. 4900 (1987)), such as enolase, glyceraldehyde-3-phosphatedehydrogenase, hexokinase, pyruvate decarboxylase, phosphofructokinase,glucose-6-phosphate isomerase, 3-phosphoglycerate mutase, pyruvatekinase, triosephosphate isomerase, phosphoglucose isomerase, andglucokinase. In constructing suitable expression plasmids, thetermination sequences associated with these genes are also ligated intothe expression vector 3' of the sequence desired to be expressed toprovide polyadenylation of the mRNA and termination. Other promoters,which have the additional advantage of transcription controlled bygrowth conditions are the promoter regions for alcohol dehydrogenase 2,isocytochrome C, acid phosphatase, degradative enzymes associated withnitrogen metabolism, and the aforementioned glyceraldehyde-3-phosphatedehydrogenase, and enzymes responsible for maltose and galactoseutilization (Holland, ibid.). Any plasmid vector containingyeast-compatible promoter, origin of relplication and terminationsequences is suitable.

In addition to microorganisms, cultures of cells derived frommulticellular organisms may also be used as hosts. In principle, anysuch cell culture is workable, whether from vertebrate or invertebrateculture. However interest has been greatest in vertebrate cells, andpropagation of vertebrate cells in culture (tissue culture) has become aroutine procedure in recent years [Tissue Culture, Academic Press, Kruseand Patterson, editors (1973)]. Examples of such useful host cell linesare VERO and HeLa cells, Chinese hamster ovary (CHO) cell lines, andW138, BHK, COS7, mouse myeloma (ATCC No. TIB 19 or TIB 20), and MDCKcell lines. Expression vectors for such cells ordinarily include (ifnecessary) an origin of replication, a promoter located in front of thegene to be expressed, along with any necessary ribosome binding sites,RNA splice sites, polyadenylation site, and transcriptional terminatorsequences.

For use in mammalian cells, the control functions on the expressionvectors are often provided by viral material. For example, commonly usedpromoters are derived from polyoma, Adenovirus 2, and most frequentlySimian Virus 40 (SV40). The early and late promoters of SV40 virus areparticularly useful because both are obtained easily from the virus as afragment which also contains the SV40 viral origin of replication(Fiers, et al, Nature, Vol. 273, pg 113 (1978) incorporated herein byreference. Smaller or larger SV40 fragments may also be used, providedthere is included the approximately 250 bp sequence extending from theHindIII site toward the Bgl I site located in the viral origin ofreplication. Further, it is also possible, and often desirable, toutilize promoter or control sequences normally associated with thedesired gene sequence, provide such control sequences are compatiblewith the host cell systems.

An origin of replication may be provided either by construction of thevector to include an exogenous origin, such as may be derived from SV40or other viral (e.g. Polyoma, Adeno, VSV, BPV, etc.) source, or may beprovided by the host cell chromosomal replication mechanism. If thevector is integrated into the host cell chromosome, the latter is oftensufficient.

In selecting a preferred host cell for transfection by the vectors ofthe invention which comprise DNA sequences encoding both t-PA and DHFRprotein, it is appropriate to select the host according to the type ofDHFR protein employed. If wild type DHFR protein is employed, it ispreferable to select a host cell which is deficient in DHFR, thuspermitting the use of the DHFR coding sequence as a marker forsuccessful transfection in selective medium which lacks hypoxanthine,glycine, and thymidine. An appropriate host cell in this case is theChinese hamster ovary (CHO) cell line deficient in DHFR activity,prepared and propagated as described by Urlaub and Chasin, Proc. Natl.Acad. Sci. (USA) Vol. 77, pg. 4216 (1980), incorporated herein byreference.

On the other hand, if DHFR protein with low binding affinity for MTX isused as the controlling sequence, it is not necessary to use DHFRresistant cells. Because the mutant DHFR is resistant to methotrexate,MTX containing media can be used as a means of selection provided thatthe host cells are themselves methotrexate sensitive. Most eukaryoticcells which are capable of absorbing MTX appear to be methotreaxatesensitive. One such useful cell line is a CHO line, CHO-K1 ATCC No. CCL61.

Invertebrate expression systems include the larvae of silk worm, Bombyxmori, infected by a baculovirus vector, BmNPV, described by Maeda etal., in Nature, Vol. 315, pgs. 892-894 (1985); and in Saibo Koguku, Vol.4, pgs. 767-779 (1985).

EXAMPLES

The following examples serve to illustrate the present invention.Selection of vectors and hosts as well as the concentration of reagents,temperatures, and the values of other variable parameters are only toexemplify application of the present invention and are not to beconsidered as limitations thereof.

EXAMPLE I De Novo Preparation of Murine IL-4 cDNAs from Cl.Ly1⁺ 2⁻ /9Cells and Transient Expression in COS 7 Monkey Cells

cDNA clones coding for IL-4 were isolated from the murine helper T cellline Cl.Ly1⁺ 2⁻ /9, which is deposited with the ATCC under accessionnumber CRL 8179 and described by Nabel et al., in Cell, Vol. 23, pgs.19-28 (1981), and in Proc. Natl. Acad. Sci., Vol. 78, pgs. 1157-1161(1981). Other murine cells known to produce BCGF activity include theEL-4 line, available from the ATCC under accession number TIB 39. Theprocedures used in this example have been disclosed in Lee et al., Proc.Natl. Acad. Sci., Vol. 83, pgs. 2061-2065 (1986), which is incorporatedby reference. Briefly, a pcD cDNA library was constructed with messengerRNA (mRNA) from concanavalin A (conA) induced Cl.Ly1⁺ 2⁻ /9 cellsfollowing the procedure of Okayama and Berg, described above. IL-3 andGM-CSF clones were eliminated from a large sublibrary of randomlyselected clones by hybridization with ³² P-labeled cDNA probes. Poolsand/or individual clones from the remainder of the sublibrary werescreened for IL-4 cDNA by transfecting COS 7 monkey cells and testingculture supernatants for MCGF and TCGF activity.

A. Induction of IL-4 Production.

Cl.Ly1⁺ 2⁻ /9 cells were induced to produce IL-4 mRNA by Con A asfollows. The cells are cultured at 5×10⁵ /ml in Dulbecco's ModifiedEagles medium (DME) with 4% heat-inactivated fetal calf serum, 5×10⁻⁵ M2-mercaptoethanol (2-ME), 2 mM glutamine, non-essential amino acids,essential vitamins and 2 μg/ml Con A. After 12-14 hrs. incubation at 37°C. in 10% CO₂, the cell suspension is centrifuged at 1500 rpm for 10minutes. The cell pellets are collected and frozen immediately at -70°C.

B. Isolation of mRNA

Total cellular DNA was isolated from cells using the guanidineisothiocyanate procedure of Chirgwin, J. et al., (Biochemistry,18:5294-5299 [1979]). Frozen cell pellets from ConA-induced Cl.Ly1⁺ 2⁻/9 cells (12 hrs after stimulation) were suspended in guanidineisothiocyanate lysis solution. Twenty ml of lysis solution was used for1.5×10⁸ cells. Pellets were resuspended by pipetting, then DNA wassheared by 4 passes through a syringe using a 16 gauge needle. Thelysate was layered on top of 20 ml of 5.7M CsCl, 10 mM EDTA in 40 mlpolyallomer centrifuge tube. This solution was centrifuged at 25,000 rpmin Beckman SW28 rotor (Beckman Instruments, Inc., Palo Alto, CA) for 40hrs at 15° C. The guanidine isothiocyanate phase containing DNA waspipetted off from the top, down to the interface. The walls of the tubeand interface were washed with 2-3 ml of guanidine isothiocyanate lysissolution. The tube was cut below the interface with scissors, and theCsCl solution was decanted. RNA pellets were washed twice with cold 70%ethanol. Pellets were then resuspended in 500 μl of 10 mM Tris.HCl pH7.4, 1 mM EDTA, 0.05% SDS. 50 μl of 3M sodium acetate was added and RNAwas precipitated with 1 ml ethanol. About 0.3 mg total RNA was collectedby centrifuging and the pellets washed once with cold ethanol.

Washed and dried total RNA pellet was resuspended in 900 μl of oligo(dT) elution buffer (10 mM Tris.HCl, pH 7.4, 1 mM EDTA, 0.5% SDS). RNAwas heated for 3 min. at 68° C. and then chilled on ice. 100 μl of 5MNaCl was added. The RNA sample was loaded onto a 1.0 ml oligo (dT)cellulose column (Type 3, Collaborative Research, Waltham, MA)equilibrated with binding buffer (10 mM Tris.HCl pH 7.4, 1 mM EDTA, 0.5MNaCl, 0.5% SDS.) Flow-through from the column was passed over the columntwice more. The column was then washed with 20 ml binding buffer. PolyA⁺mRNA was collected by washing with elution buffer. RNA usually eluted inthe first 2 ml of elution buffer. RNA was precipitated with 0.1 volume3M sodium acetate (pH 6) and two volumes of ethanol. The RNA pellet wascollected by centrifugation, washed twice with cold ethanol, and dried.The pellet was then resuspended in water. Aliquots were diluted, andabsorbance at 260 nm was determined.

C. Construction of pcD cDNA Library

(1) Preparation of Vector Primer and Oligo(dG)-Tailed Linker DNAs.

The procedure of Okayama & Berg (Mol. & Cell. Biol. Voil. 2, pgs.161-170 [1982]) was used with only minor modifications. The pcDVl andpLl plasmids are described by Okayama & Berg (Mol. & Cell. Biol.3:380-389 [1983]) and are available from Pharmacia (Piscataway, N.J.).Specifically, a modified pcDVl plasmid was used which contained an NsiIsite at the previous location of the KpnI site.

An 80 μg sample of pcDVl DNA was digested at 30° C. with 20 U of KpnIendonuclease in a reaction mixture of 450 μl containing 6 mM Tris.HCl(pH 7.5), 6 mM MgCl₂, 6 mM NaCl, 6 mM 2-ME, and 0.1 mg of bovine serumalbumin (BSA) per ml. After 16 hr the digestion was terminated with 40μl of 0.25M EDTA (pH 8.0) and 20 μl of 10% sodium dodecyl sulfate (SDS);the DNA was recovered after extraction with water-saturated 1:1phenol-CHCl₃ (hereafter referred to as phenol-CHCl₃) and ethanolprecipitation. Homopolymer tails averaging 60, but not more than 80,deoxythymidylate (dT) residues per end were added to the NsiIendonuclease-generated termini with calf thymus terminal transferase asfollows: The reaction mixture (38 μl) contained sodium cacodylate-30 mMTris.HCl pH 6.8 as buffer, with 1 mM CoCl₂, 0.1 mM dithiothreitol, 0.25mM dTTP, the NsiI endonuclease-digested DNA, and 68 U of the terminaldeoxynucleotidyl transferase (P-L Biochemicals, Inc., Milwauke, WI).After 30 min. at 37° C. the reaction was stopped with 20 μl of 0.25MEDTA (pH 8.0) and 10 μl of 10% SDS, and the DNA was recovered afterseveral extractions with phenol-CHCl₃ by ethanol precipitation. The DNAwas then digested with 15 U of EcoRI endonuclease in 50 μl containing 10mM Tris.HCl pH 7.4, 10 mM MgCl₂, 1 mM dithiothreitol, and 0.1 mg of BSAper ml for 5 hr at 37° C. The large fragment, containing the SV40polydenylation site and the pBR322 origin of replication andampicillin-resistance gene, was purified by agarose (1%) gelelectrophoresis and recovered from the gel by a modification of theglass powder method (Vogelstein, B. & Gillespie, D., Proc. Natl. Acad.Sci. 76: 615-619 [1979]). The dT-tailed DNA was further purified byabsorption and elution from an oligo (dA)-cellulose column as follows:The DNA was dissolved in 1 ml of 10 mM Tris.HCl pH 7.3 buffer containing1 mM EDTA and 1M NaCl, cooled at 0° C., and applied to an oligo(dA)-cellulose column (0.6 by 2.5 cm) equilibrated with the same bufferat 0° C. and eluted with water at room temperature. The eluted DNA wasprecipitated with ethanol and dissolved in 10 mM Tris.HCl pH 7.3 with 1mM EDTA.

The oligo (dG) tailed linked DNA was prepared by digesting 75 μg of pLlDNA with 20 U of PstI endonuclease in 450 μl containing 6 mM Tris.HCl pH7.4, 6 mM MgCl₂, 6 mM 2-ME, 50 mM NaCl, and 0.01 mg of BSA per ml. After16 hr at 30° C. the reaction mixture was extracted with phenol-CHCl₃ andthe DNA was precipitated with alcohol. Tails of 10 to 15 deoxyguanylate(dG) residues were then added per end with 46 U of terminaldeoxynucleotidyl transferase in the same reaction mixture (38 μl) asdescribed above, except that 0.1 mM dGTP replaced dTTP. After 20 min. at37° C. the mixture was extracted with phenol-CHCl₃, and after the DNAwas precipitated with ethanol it was digested with 35 U of HindIIIendonuclease in 50 μl containing 20 mM Tris.HCl pH 7.4, 7 mM MgCl.sub.2, 60 mM NaCl, and 0.1 mg of BSA at 37° C. for 4 hr. The small oligo(dG)-tailed linker DNA was purified by agarose gel (1.8%)electrophoresis and recovered as described above.

(2) cDNA Library Preparation:

Step 1: cDNA synthesis. The reaction mixture (10 μl) contained 50 mMTris.HCl pH 8.3, 8 mM MgCl₂, 30 mM KCl, 0.3 mM dithiothreitol, 2 mM eachdATP, dTTP, dGTP, and dCTP, 20 μCi ³² P-dCTP (3000 Ci/mmole), 3 μgpolyA⁺ RNA from Con-A induced T-cells, 60 units RNasin (a tradenamedribonuclease inhibitor from Promega Biotec, Inc., Madison, WI), and 2 μgof the vector-primer DNA (15 pmol of primer end), and 45 U of reversetranscriptase. The reaction was incubated 60 min at 42° C. and thenstopped by the addition of 1 μl of 0.25M ETDA (pH 8.0) and 0.5 μl of 10%SDS; 40 μl of phenol-CHCl₃ was added, and the solution was blendedvigorously in a Vortex mixer and then centrifuged. After adding 40 μl of4M ammonium acetate and 160 μl of ethanol to the aqueous phase, thesolution was chilled with dry ice for 15 min., warmed to roomtemperature with gentle shaking to dissolve unreacted deoxynucleosidetriphosphates that had precipitated during chilling, and centrifuged for10 min. in an Eppendorf microfuge. The pellet was dissolved in 10 μl of10 mM Tris.HCl pH 7.3 and 1 mM EDTA, mixed with 10 μl of 4M ammoniumacetate, and reprecipitated with 40 μl of ethanol, a procedure whichremoves more than 99% of unreacted deoxynucleotide triphosphates. Thepellet was rinsed with ethanol.

Step 2: Oligodeoxycytidylate [oligo (dC)] addition. The pelletcontaining the plasmid-cDNA:mRNA was dissolved in 20 μl of 140 mM sodiumcacodylate-30 mM Tris.HCl pH 6.8 buffer containing 1 mM CoCl₂, 0.1 mMdithiothreitol, 0.2 μg of poly (A), 70 μM dCTP, 5 μCi ³² P-dCTP, 3000Ci/mmole, and 60 U of terminal deoxynucleotidyl transferease. Thereaction was carried out at 37° C. for 5 min. to permit the addition of10 to 15 residues of dCMP per end and then terminated with 2 μl of 0.25MEDTA (pH 8.0) and 1 μl of 10% SDS. After extraction with 20 μl ofphenol-CHCl₃, the aqueous phase was mixed with 20 μl of 4M ammoniumacetate, the DNA was precipitated and reprecipitated with 80 μl ofethanol, and the final pellet was rinsed with ethanol.

Step 3: HindIII endonuclease digestion. The pellet was dissolved in 30μl of buffer containing 20 mM Tris.HCl pH 7.4, 7 mM MgCl₂, 60 mM NaCl,and 0.1 mg of BSA per ml and then digested with 10 U of HindIIIendonuclease for 2 hr at 37° C. The reaction was terminated with 3 μl of0.25M EDTA (pH 8.0) and 1.5 μl of 10% SDS and, after extraction withphenol-CHCl₃ followed by the addition of 30 μl of 4M ammonium acetate,the DNA was precipitated with 120 μl of ethanol. The pellet was rinsedwith ethanol and then dissolved in 10 μl of 10 mM Tris.HCl (pH 7.3) and1 mM EDTA, and 3 μl of ethanol was added to prevent freezing duringstorage at -20° C.

Step 4: Cyclization mediated by the oligo (dG)-tailed linker DNA. A 9 μlsample of the HindIII endonuclease-digested oligo (dC)-tailed cDNA:mRNAplasmid (about 90% of the sample) was incubated in a mixture (90 μl)containing 10 mM Tris.HCl pH 7.5, 1 mM EDTA, 0.1M NaCl, and 1.8 pmol ofthe oligo (dG)-tailed linker DNA at 65° C. for 5 min., shifted to 42° C.for 60 min, and then cooled to 0° C. The mixture (90 μl) was adjusted toa volume of 900 μl containing 20 mM Tris.HCl pH 7.5, 4 mM MgCl₂, 10 mM(NH₄)₂ SO₄, 0.1M KCl, 50 μg of BSA per ml, and 0.1 mM β-NAD; 6 μg of E.coli DNA ligase were added and the solution was then incubated overnightat 12° C.

Step 5: Replacement of RNA strand by DNA. To replace the RNA strand ofthe insert, the ligation mixture was adjusted to contain 40 μM of eachof the four deoxynucleoside triphosphates, 0.15 mM beta-NAD, 4 μg ofadditional E. coli DNA ligase, 16 U of E. coli DNA polymerase I (PolI,)and 9 U of E. coli RNase H. This mixture (960 μl) was incubatedsuccessively at 12° C. and at room temperature for 1 hr each to promoteoptimal repair synthesis and nick translation by PolI.

Step 6: Transformation of E. coli. Transformation was carried out usingminor modifications of the procedure described by Cohen et al. (Proc.Nat. Acad. Sci. U.S.A., 69:2110-2114 [1972]). E. coli K-12 strain MC1061(Casadaban, M. and Cohen, S., J. Mol. Biol. 138:179-207 [1980]) wasgrown to 0.5 absorbancy unit at 600 nm at 37° C. in 300 ml of L-broth.The cells were collected by centrifugation, suspended in 30 ml of 10 mMPipes. pH 7, 60 mM CaCl₂, 15% glycerol and centrifuged at 0° C. for 5min. The cells were resuspended in 24 ml of the above buffer andincubated again at 0° C. for 5 min.; then, 1.0 ml aliquots of the cellsuspensions were mixed with 0.1 ml of the DNA solution (step 5) andincubated at 0° C. for 20 min. Next the cells were kept at 42° C. for 2min. and thereafter at room temperature for 10 min.; then 1 liter ofL-broth was added, and the culture was incubated at 37° C. for 60 min.Ampicillin was added to a concentration of 50 μg/ml. The culture wasshaken for an additional 10 hrs. at 37° C. Dilutions of this culturewere spread on L-broth agar containing 50 μg/ml ampicillin. Afterincubation at 37° C. for 12 to 24 hr, individual colonies were pickedwith sterile tooth-picks. In all, approximately 1×10⁵ independent cDNAclones were generated.

D. Screening the pcD Library.

10⁴ single clones were picked at random from the T-cell cDNA library andpropagated individually in wells of microtiter dishes containing 200 μlL-broth with ampicillin at 50 μg/ml and dimethyl sulfoxide at 7%. Tofocus only on the novel MCGF activity, 53 IL-3 cDNA clones and oneGM-CSF cDNA clone identified by hybridization with the appropriate ³²P-labelled cDNA probes constructed from the clones disclosed by Lee etal., Proc. Natl. Acad. Sci., Vol. 82, pgs. 4360-4364 (1985); and Yokotaet al., Proc. Natl. Acad. Sci., Vol. 81, pgs. 1070-1074 (1984). Theprocedure was carried out as follows: Each plate of 96 cultures wasreplicated onto nitrocellulose filters for hybridization screening.Hybridizations were performed in 6XSSPE (1XSSPE=180 mM NaCl; 10 mMsodium phosphate, pH 7.4; 1 mm EDTA), 0.1% SDS, 100 μg/ml E. coli tRNA,50% formamide, for 16 hrs. at 42° C. Hybridizing clones were identifiedby autoradiography of the washed filter. These clones were removed bysterilizing the microtiter wells containing these clones with ethanolprior to the preparation of clone pools. Pools containing up to 48 cDNAclones were prepared from the microtiter cultures. Two hundred suchpools were grown up in 1 liter cultures of L-broth containing 100 μg/mlampicillin. Plasmid DNA was isolated from each culture and purified bytwice banding through CsCl gradients. The DNA representing each pool wastransfected into COS7 monkey cells as follows. (COS7 cells are describedby Gluzman in Cell, Vol. 23, pgs. 175-180 (1981), and are available fromthe ATCC under accession number CRL 1651).

One day prior to transfection, approximately 10⁶ COS 7 monkey cells wereseeded onto individual 100 mm plates in DME containing 10% fetal calfserum and 2 mM glutamine. To perform the transfection, the medium wasaspirated from each plate and replaced with 4 ml of DME containing 50 mMTris.HCl pH 7.4, 400 μg/ml DEAE-Dextran and 50 μg of the plasmid DNAs tobe tested. The plates were incubated for four hours at 37° C., then theDNA-containing medium was removed, and the plates were washed twice with5 ml of serum-free DME. DME containing 150 μM Chloroquine was added backto the plates which were then incubated for an additional 3 hrs at 37°C. The plates were washed once with DME and then DME containing 4% fetalcalf serum, 2 mM glutamine, penicillin and streptomycin was added. Thecells were then incubated for 72 hrs at 37° C. The growth medium wascollected and evaluated in the various bioassays.

An initial set of plasmid pools was screened primarily by usingproliferation assays for TCGF and MCGF activities with the HT-2(described more fully below) and MC/9 cell lines, respectively. Amongthe first 110 pools assayed on these two cell lines, eight producedsignificant activity in the HT-2 TCGF assay. Several of these pools hadweak but significant MCGF activity, but because the MCGF activities weregenerally weaker and more variable, we did not rely on this assay foridentifying positive pools.

Approximately half of the COS supernatants from the random pooltransfections were also assayed for Ia inducing activity on mouse Bcells. Among the pools tested, each pool shown to be active for TCGFactivity was found also to have Ia inducing activity. Thus, there was aperfect correlation between the TCGF activity and the Ia inducingactivity.

One pool, 2A, which was reproducibly the most active in all assays, wassubdivided into 48 smaller subpools. Two subpools were positive for bothMCGF and TCGF activities. The single clone, 2A-E3, common to bothsubpools was then grown individually and its plasmid DNA was transfectedinto COS 7 cells as described above. The resulting COS supernatant wasthen assayed for the presence of various activities, including MCGF,TCGF, Ia inducing, and IgE and IgG enhancing activities.

A 366 base-pair-long PstI fragment isolated from clone 2A-E3 (FIG. 1A)and labelled with ³² P was used as a probe to screen pools which hadbeen positive for biological activity as well as other untested pools.The screening was performed by hybridization to filters replicated withthe microtiter cultures as described above. Nine hybridizing clones wereisolated and their DNA analyzed by restriction mapping. All pools whichexhibited biological activity contained at least one hybridizing clonewhich shared a common restriction cleavage map with clone 2A-E3. Thefrequency of hybridizing clones among the 10⁴ which were picked suggestsa frequency of approximately 0.2% in the total library. Of thehybridizing clones which were tested, approximately 90% expressed afunctional protein.

E. Biological Activities of Culture Supernatants of COS 7 Monkey CellsTransfected with pcD-2A-E3.

Supernatant from COS 7 cells transfected with pcD-2A-E3 was tested forTCGF activity on the murine helper T cell line HT-2, described by Watsonin J. Exp. Med., Vol. 150, pg. 1510 (1979). Proliferation of the HT-2cells, as determined by the colorimetric assay of Mosmann (cited above),was used as a measure of TCGF activity (degree of proliferation beingcorrelated to optical density (OD) between 570-630 nm). FIG. 3Aillustrates the relative TCGF activities at various dilutions of (i)supernatant from COS 7 cells transfected with pcD-2A-E3 (curve 1), (ii)supernatant from Cl.Ly1⁺ 2⁻ /9 cultures (curve 2), (iii) supernatantfrom COS 7 cells transfected with a pcD plasmid carrying IL-2 cDNA(curve 3), and (iv) supernatant from COS 7 cells transfected with a pcDplasmid containing no cDNA insert (i.e. a "mock" transfection) (curve4).

Similarly, supernatants from pcD-2A-E3 transfected COS 7 cells weretested for MCGF activity on MC/9 cells, again using the colorimetricassay of Mosmann to measure MC/9 proliferation. FIG. 3B illustratesrelative MCGF activity of (i) supernatant from COS 7 cells transfectedwith pcD-2A-E3 (curve 1), (ii) supernatant from COS 7 cells transfectedwith a pcD plasmid carrying IL-3 cDNA (curve 2), (iii) supernatant fromCl.Ly1⁺ 2⁻ /9 cells (curve 3), and (iv) supernatant from mocktransfected COS 7 cells (curve 4).

FIG. 3C illustrates the results of an Ia induction assay conducted on(i) supernatant of COS 7 cells transfected with pcD-2-E3 (curve 1), (ii)supernatants of Cl.Ly1⁺ 2⁻ /9 cells (curve 2), and (iii) supernatants ofmock transfected COS 7 cells (curve 3). The Ia induction assay wascarried out as described by Roehm et al. (cited above). Several DBA/2mice (2-3 months old) were sacrificed and the spleens obtainedsurgically. The erythrocytes were lysed by hypotonic shock using 0.87%ammonium chloride. Then the T-cells were lysed by using cytotoxicmonoclonal antibodies directed against T-cell-specific surface markers(Thy-1, Lyt-1 and Lyt-2) followed by incubation in rabbit complement.The dead cells were then removed using ficollhypaque density gradients.Adherent cells had been removed previously by adherence to plastic petridishes at 37° C. At this time the cells were washed, counted and scoredfor viability. Approximately one million cells were incubated in 0.5 mlof tissue culture medium (RPMI 1640 or Minimal essentialmedium-MEM/Earle's salts) (Gibco) supplemented with 10% fetal calfserum, 2-mercaptoethanol and various antibiotics (penicillin,streptomycin and gentamicin). In experiments where the positive controlconsisted of supernatants from T-cells induced with the T-cell mitogenConcanavalin A, 10 mg/ml (final concentration) of alpha-methyl mannosidewas added to neutralize the mitogen. After 24 hours incubation, thecells were harvested and prepared for staining with anti-I-A^(d) oranti-I-A^(bd) monoclonal antibodies. These antibodies were used as firststage antibodies conjugated to either the hapten N.I.P. or biotin. Thestaining was then completed by incubating the cells with fluoresceinatedsecond-stage reagents (either anti-NIP antibodies or avidin). Theintensity of fluorescence staining was then determined using either afluorescence-activated cell sorter (Becton-Dickinson, Mountain View, CA)or a Cytofluorograph (Ortho Diagnostics, Cambridge, MA). Fluorescenceunits in FIG. 3C are calculated by multiplying the percentage ofpositive cells in each sample by the intensity of fluorescent staining.

FIG. 3D graphically illustrates the degrees by which IgE and IgG₁production are induced in T cell depleted mouse spleen cells by (i) COS7 medium alone (bar 1), (ii) 20% supernatant from mock transfected COS 7cells (bar 2), (iii) 10% supernatant from Cl.Ly1⁺ 2⁻ /9 cells plus 20%supernatant from mock transfected COS 7 cells (bar 3), and (iv) 20%supernatant from pcD-2A-E3 transfected COS 7 cells (bar 4). Levels ofIgE and IgG₁ were determined by the isotype-specific ELISA describedabove.

Murine IL-4 was found to enhance the MCGF activity of IL-3 in MC/9cells, Smith and Rennick, Proc. Natl. Acad. Sci., Vol. 83, pgs.1857-1861 (1986). And Murine IL-4 was found to enhance GM-CSF stimulatedproliferation of the IL-3 dependent cell line, NFS-60, described byHolmes et al., in Proc. Natl. Acad. Sci., Vol. 82, pgs. 6687-6691(1985).

F. Structure of pcD-2A-E3 and Nucleotide Sequence of Its cDNA Insert.

The structure of pcD-2A-E3 is illustrated diagramatically in FIG. 2A,and an expanded restriction map of its insert is illustrated in FIG. 2B.The insert was sequenced using both the Maxam and Gilbert approach(Methods in Enzymology, Vol. 65, pgs. 499-560 (1980)) and the Sangerapproach (Proc. Natl. Acad. Sci., Vol. 74, pgs. 5463-5467 (1977)). Thesequence is illustrated in FIG. 1A, along with the deduced amino acidsequence for the longest open reading frame in-phase with the first ATGstart codon. The single long open reading frame in the mouse 2A-E3 cDNAclone consists of 140 amino acid residues. Because this lymphokine is asecreted protein, a hydrophobic leader sequence would be expected toprecede the sequence for the mature secreted form of the protein.Analysis of the hydrophobicity of the polypeptide and comparison with aproposed consensus sequence for the processing of signal peptides(Perlman et al., J. Mol. Biol., Vol. 167, pgs. 391-409 (1983)) suggestthat cleavage of the precursor polypeptide would occur following theserine residue at amino acid position 20 in FIG. 1A. Grabstein et al.,J. Exp. Med., Vol. 163, pgs. 1405-1414 (1986), has confirmed that theN-terminal sequence of secreted murine IL-4 begins at the position 21His of FIG. 1A.

EXAMPLE II Preparation of Human IL-4 Via a Murine cDNA Probe to a HumanHelper T Cell cDNA Library and Transient Expression in COS 7 MonkeyCells and Mouse L Cells

cDNA clones coding for IL-4 were isolated from cDNA librariesconstructed from an induced human helper T cell, 2F1, and induced humanperipheral blood lymphocytes (PBLs) by way of a murine cDNA probe. Otherhuman cell lines known to produce BCGF activity include variants of theCEM line, available from the ATCC under accession numbers CCL 119, CRL8436, and TIB 195, and described by Foley et al., in Cancer, Vol. 18,pgs. 522-529 (1965), and by Ligler, in Lymphokine Research, Vol. 3, pgs.183-191 (1984). The procedures used in this example are disclosed byYokota et al., in Proc. Natl. Acad. Sci., Vol. 83, pgs. 5894-5898(1986), which is incorporated herein by reference.

A human helper T-cell clone, 2F1, and human PBLs were grown in Iscove'smedium supplemented with 3% fetal calf serum. The 2F1 cells wereactivated with Con A (10 μg/ml) and PBL's were stimulated with 1 ng/mlPMA for 12 hrs, after which Con A at 5 μg/ml was added. The cells wereharvested 4 hr (2F1) or 10 hr (PBL's) after addition of Con A.

mRNA extraction and cDNA library construction were carried out asdescribed in Example I. A PstI fragment was isolated from the mousepcD-2A-E3 cDNA clone, labeled by nick translation (1×10⁸ cpm/μg) andused to probe nitrocellulose filters containing plasmid DNA preparationsfrom ten pools, each representing approximately 1×10³ clones of 2F1 cDNAlibrary. Low stringency hydridization conditions (overnight at 42° C.)were used: 6xSSPE (1×SSPE=180 mM NaCl/10 mM sodium phosphate, pH 7.4/1mM EDTA) (Maniatis, T. et al., Molecular Cloning: A Laboratory Manual(Cold Spring Harbor Laboratory, N.Y., 1982)), 20% (vol/vol) formamide,0.1% sodium dodecyl sulfate, yeast carrier tRNA at 100 μl. The filterswere washed with 2×SSPE, 0.1% sodium dodecyl sulfate at 37° C.

A single clone (pcD-46) was identified in one of the ten pools.Additional clones were obtained by screening the PBL cDNA libraries witha probe constructed from the NheI-EcoRI fragment of pcD-46 (illustratedin the restriction map of FIG. 2D). Analysis by restriction enzymesindicated that the PBL clones were identical in structure to pcD-46.

It was discovered that a guanidine-rich region in the 5', or upstream,direction from the coding region insert pcD-46 inhibited expression ofthe IL-4 polypeptide. Consequently, the insert of pcD-46 was recloned toremove the guanidine-rich region. The resulting clone is designatedpcD-125. It was also discovered that expression was improved bytransfecting mouse L cells with pcD-46.

The vector pcD-125 was formed as follows: pcD-46 was cleaved with Sau3Ato isolate a fragment containing the 5' 162 nucleotides of the cDNAinsert (eliminating the GC segment) and then the fragment was insertedinto the BglII site of p101. The plasmid p101 was derived from pcD-mouseIL-3 (see, Yokota, T. et al., [1984]cited above) and is deleted for thesequence from the PstI site at the 5' end of the cDNA to a BglII sitewithin the mouse IL-3 cDNA. A BglII site is included at the junction ofthe deleted sequence. The Sau3A fragment is fused to the SV40 promoteras in pcD-46, except for the GC stretch. The remainder of the human cDNAwas then reconstructed with a HindIII/NheI fragment from pcD-46 whichcarries the 3' end of the cDNA, the SV40 poly A site and all of thepBR322 sequences of pcD-46.

Supernatants of the pcD-46 and pcD-125 transfected COS 7 and L cellswere assayed for BCGF and TCGF activity. TCGF was assayed with anEpstein-Barr virus transformed human helper T cell line JL-EBV, andphytohemagglutinin (PHA) stimulated human peripheral blood lymphocytes(PBLs).

The human helper T-cell clone JL-EBV was stimulated with irradiated(4500R) cells of a human EBV-transformed B-cell line, and subsequentlymaintained in RPMI 1640 medium containing 10% human AB serum, 50micromolar 2-mercaptoethanol (2ME) and recombinant human IL-2. HumanPBLs were stimulated with PHA (20 microgram/ml) and maintained in RPMI1640 containing 10% fetal calf serum, 50 micromolar 2ME and recombinanthuman IL-2. Five to ten days after stimulation, JL-EBV cells or PHAblasts were used as targets in a two-day TCGF assay, using the Mosmanncolorimetric method (described above) or in a three-day TCGF assay,using [³ H] thymidine incorporation.

FIG. 4A illustrates the TCGF activities measured by JL-EBV cells(colorimetric assay) of (i) supernatant from COS 7 cells transfectedwith pcD plasmids expressing human IL-2 (curve A); (ii) supernatant fromL cells transfected with pcD-125 (curve B); (iii) supernatant from COS 7cells transfected with pcD-125 (curve C), (iv) supernatant from COS 7cells transfected with pcD-46 (curve D), and (v) supernatant from mocktransfected COS 7 cells (curve E). FIG. 4B illustrates the TCGFactivities measured by PHA stimulated PBLs (colorimetric assay) of (i)supernatant from COS 7 cells transfected with pcD plasmids expressinghuman IL-2 (curve A), (ii) supernatant from COS 7 cells transfected withpcD-125 (curve B), and (iii) supernatant from mock transfected COS 7cells (curve C). FIG. 4C illustrates the TCGF activities measured by PHAstimulated PBLs (tritiated thymidine incorporation assay) of (i)supernatant from COS 7 cells transfected with pcD-125 (curve A), (ii)supernatant from COS 7 cells transfected with pcD plasmids expressinghuman IL-2 (curve B), and (iii) supernatant from mock transfected COS 7cells (curve C).

BCGF activity of various dilutions of pcD-125 transfection supernatantswere compared with the BCGF activity of a BCGF ("commercial BCGF")described by Maizel et al., Proc. Natl. Acad. Sci,. Vol. 79, pgs.5998-6002 (1982), and commercially available from Cytokine TechnologyInternational (Buffalo, NY). Table IV illustrates the BCGF activities ofvarious dilutions of COS 7 transfection supernatants on anti-IgMantibody preactivated B cells. B cells were prepared as described in theassay section above.

                  TABLE IV                                                        ______________________________________                                        Effect of the IL-4 cDNA transfection super-                                   natants on anti-IgM-preactivated B cells                                      (vol/vol)                                                                             .sup.3 H-Thymidine Incorporation (cpm)                                of super-                 Mock-                                               natants Mock-     Clone   transfection +                                                                          Cone 125 +                                added   transfection                                                                            125     10% BCGF  10% BCGF                                  ______________________________________                                        0       278        278    1835      1835                                      0.2     189        144    1362      2303                                      1       323       1313    1699      3784                                      5       408       4314    1518      7921                                      15      397       4289    1093      8487                                      ______________________________________                                    

Table V illustrates the BCGF activities of various dilutions of COS 7transfection supernatants on SAC preactivated B cells (prepared asdescribed above).

                  TABLE V                                                         ______________________________________                                        Activity of the IL-4 cDNA transfection                                        supernatants on SAC-preactivated B cells                                      (vol/vol)                                                                             .sup.3 H-Thymidine Incorporation (cpm)                                of super-                 Mock-                                               natants Mock-     Clone   transfection +                                                                          Cone 125 +                                added   transfection                                                                            125     10% BCGF  10% BCGF                                  ______________________________________                                        0       2237      2237    12,992    12,992                                    0.2     1789      2682    13,126     5,655                                    1        740      2374    13,714     6,765                                    5       1285      2826     5,848    10,023                                    15      1560      4701    10,128    10,924                                    ______________________________________                                    

Although the human IL-4 of the invention and commercial BCGF bothdisplay BCGF activity, Mehta et al., in J. Immunol., Vol. 135, pgs.3298-3302 (1985), demonstrated that TCGF activity can be biochemicallyseparated from the BCGF activity of commercial BCGF, indicating that theactivities are caused by separate molecules. Thus, human IL-4 andcommercial BCGF are different molecules because TCGF activity isinseparable from BCGF activity in human IL-4, using standard biochemicalfractionation techniques.

Supernatants from COS-7 cells transfected with plasmids bearing thehuman IL-4 cDNA induce the proliferation of normal human T cells and thehuman T-cell clone JL-EBV, and activity that is similar to mouse IL-4.However, the maximum extent of proliferation of human T cells induced inresponse to human IL-4 is about half of that induced by human IL-2. Theproliferation-inducing activity of IL-4 could not be inhibited bymonoclonal antibodies against IL-2 or the IL-2 receptor when tested.These results suggest that IL-4 acts directly on T cells and not by wayof the induction of IL-2 and that its activity is not mediated by theIL-2 receptor. The COS-human IL-4 supernatants also stimulate theproliferation of human B cells preactivated with optimal concentrationsof anti-IgM antibodies coupled to beads and have additive proliferativecapacity with commercial BCGF at saturation levels of the BCGF assay.This suggests that human IL-4 and commercial BCGF operate on B cells bydifferent routes, e.g. possibly by different sets of receptors. Thesupernatants did not significantly induce proliferation of B cellspreactivated with SAC, whereas commercial BCGF purified fromsupernatants of PBL cultures stimulated with PHA strongly induced theproliferation of SAC-preactivated human B cells. These results furtherindicate that the human IL-4 cDNA encodes a BCGF activity that isdistinct from that present in the commercial BCGF.

Supernatant of pcD-125 transfected COS 7 cells was also tested for itsability to induce Fc-epsilon receptors on tonsilar B cells. Human tonsilcells were dispersed into a single cell suspension using standardtechniques. The B cell population was enriched using the protocoldescribed above, and the enriched cells were stimulated with anti-IgMantibody for 24 hours in culture medium at 37° C. Fc-epsilon receptorbearing cells were assayed by a Becton Dickinson FACS IV cell sorterusing a fluorescently labeled monoclonal antibody specific for thereceptor using the technique disclosed by Bonnefoy et al., in J.Immunol. Meth., Vol. 88, pgs. 25-32 (1986). FIGS. 5A-5D are histogramsillustrating cell frequency (ordinate) versus fluorescent intensity(abscissa). Fluorescence intensity is proportional to the number ofFc-epsilon receptors present on a cell. In all the Figures the cellshave been stimulated with anti-IgM. FIGS. 5A through 5D correspond toexposures to media consisting of 0%, 0.1%, 1%, and 10% supernatant frompcD-125 transfected COS 7 cells.

The DNA sequence of the cDNA insert of clone #46 was determined and isshown in FIG. 1B. The cDNA insert is 615 bp long, excluding the poly(A)tail. There is a single open reading frame, with the first ATG codonlocated at 64 nucleotides from the 5' end followed by 153 codons endingwith the termination codon TAG at nucleotide positions 523-525. TheN-terminal segment of the predicted polypeptide is hydrophobic, as wouldbe expected for a secreted protein.

A comparison between the coding regions of a human and a mouse cDNA ofthe present invention revealed that the regions of the human cDNA codingsequence in pcD-46 covered by amino acid positions 1-90 and 129-149share approximately 50% homology with the corresponding regions of themouse cDNA (2A-E3) coding sequence. These regions, and 5' and 3'untranslated regions, share about 70% homology between the two cDNAsequences from the different species, whereas the region covered byamino acids 91-128 of the human protein shares very limited homologywith the corresponding mouse region. In all, six of the seven cysteineresidues in the human protein are conserved in the related mouseprotein. Some amino acid sequence homology exists between a native formof a human polypeptide of the present invention and mouse IL-3. Aminoacid residues 7-16 and 120-127 are 50% and 55% homologous, respectively,to residues 16-27 and 41-49 of the mouse IL-3 precursor polypeptide(Yokota, T. et al., Proc. Natl. Acad. Sci. U.S.A. 81:1070-1074 [1984]).

As described more fully below, human IL-4 purified from pcD-125transfected COS 7 supernatants was found to be the 129 amino acidpolypeptide having the sequence illustrated by FIG. 1C.

EXAMPLE III Enhanced Expression of Human IL-4 in COS 7 Monkey cells byUsing an Epstein-Barr Virus (EBV) Derived Vector Containing an RSV-LTRPromoter

A 10-20 fold enhancement of human IL-4 expression was obtained byrecloning the XhoI fragment of pcD-125 into an EBV-derived vectorcontaining a Rous sarcoma virus long terminal repeat (RSV-LTR) promoter.The EBV-derived vector and the RSV-LTR promoter are described in thefollowing references, which are incorporated by reference: Gorman etal., Proc. Nat. Acad. Sci., Vol. 79, pgs. 6777-6781 (1982); and Yates etal., Nature, Vol. 313, pgs. 812-815 (1985).

A HindIII/XhoI fragment containing the RSV-LTR promoter was isolatedfrom a pcD plasmid previously constructed from the RSV-LTR containingAccI/HindIII fragment described by Gorman et al. (cited above) and acommercially available pcD vector (e.g. Pharmacia). The aboveHindIII/XhoI fragment and a HindIII/XhoI fragment from a pL1 plasmid(Pharmacia) containing an SV40 origin of replication (ori) are splicedinto plasmid pcDV1 (available from Pharmacia), the orientation of theSV40 ori region not being critical. Between an AatII site and an NdeIsite, the resulting pcD vector contains in sequence (from the AatIIsite) an SV40 ori region, an RSV-LTR promoter, and the SV40 poly Aregion. After the XhoI fragment of pcD-125 is isolated and inserted intothe XhoI site of the just constructed pcD vector, the unique AatII andNdeI sites on the vector are converted into SalI sites using standardtechniques. Briefly, the pcD vector is digested with AatII and NdeI, theIL-4 containing fragment is isolated, and the isolated fragment istreated with T4 DNA polymerase in the presence of appropriateconcentrations of the nucleoside triphosphates. The 5'→3' DNA polymeraseactivity of T4 DNA polymerase fills in the 5' protruding ends of therestriction cuts, and the 3'→5' exonuclease activity of T4 DNApolymerase digests the 3' protruding ends of the restriction cuts toleave a blunt ended fragment, to which kinased SalI linkers (New EnglandBiolabs) are ligated using T4 DNA ligase.

The above SalI fragment (illustrated in FIG. 11) is inserted in theEBV-derived vector p201 described by Yates et al. (cited above) at thelocation of a unique ClaI site, which had been converted to a SalI siteusing standard techniques. Briefly, p201 (illustrated in FIG. 11) isdigested with ClaI and treated with DNA polymerase I (Klenow fragment)and appropriate concentrations of nucleoside triphosphates. Thisprocedure fills in the protruding ends of the ClaI cut to leave a bluntended fragment. Next, the blunt ends are ligated to a kinased SalIlinker. The resulting EBV-derived vector containing the RSV-LTR promoterand human IL-4 cDNA insert is referred to herein as pEBV-178.

pEBV-178 was transfected into COS 7 cells using standard techniques andthe culture supernatants were assayed for TCGF activity as a measure ofIL-4 expression.

EXAMPLE IV Expression of Native Human IL-4 and Mutein IS^(O)(Ala-Glu-Phe) in E. coli

Two vectors containing human IL-4 cDNA inserts were constructed forexpression of human IL-4 in E. coli: a pIN-III secretion vector whichcontains the signal peptide sequence of the ompA protein("pIN-III-ompA2"), and a pUC12 plasmid containing a trpP promoter and anadjacent ribosome binding site (RBS) region ("TRPC11").

A. pIN-III-ompA2

Two vectors were constructed using the pIN-III-ompA2 plasmid, which isdescribed by Ghrayeb et al., in EMBO Journal, Vol. 3, pgs. 2437-2442(1984); and Masui et al., in Biotechnology, Vol. 2, pgs. 81-85 (1984).Accordingly, these references are incorporated by reference.

The first vector, designated pIN-III-ompA2(1), was constructed byligating, in series, the EcoRI/BAMHI digested pIN-III-ompA2 plasmid, asynthetic linker, and the BamHI/EcoRV fragment of pcD-125. The syntheticlinker used in this construction resulted in the secretion of abiologically active IL-4 polypeptide having the three extra N-terminalamino acids Ala-Glu-Phe- (i.e. mutein IL^(O) (Ala-Glu-Phe) wassecreted). The synthetic linker consisted of the following sequences ofnucleotides:

    ______________________________________                                        AA      TTC         CAC   AAG      TGC   GAT                                          G           GTG   TTC      ACG   CTA                                  ______________________________________                                    

EcoRI/BamHI digested pIN-III-ompA2 and the BamHI/EcoRV fragment ofpcD-125 were mixed in a standard ligation solution (e.g. Maniatis etal., cited above) containing 0.1 micromolar of the synthetic linker. E.coli strain Ab1899 was infected by the pIN-III-ompA2(1) plasmid andtransformants were selected by colony hybridization using a ³² P-labeledIL-4 cDNA probe. Human IL-4 extracts for assaying were obtained asfollows. After sonication, the bacterial cultures were centrifuged, andthe supernatant removed from the pellet. The pellet was treated with 1%SDS, 2 mM dithiothreitol, and 6M guanidine. The material wasrecentrifuged, the supernatant discarded, and the pellet treated at 45°C. with 3% SDS and 2 mM dithiothreitol. The material was againcentrifuged, and the supernatant assayed by SDS-PAGE.

pIN-III-ompA(2) was constructed so that the native human IL-4 would beexpressed. The three amino acid addition in the pIN-III-ompA(1)construction was eliminated by site-specific mutagenesis of the ompAsignal peptide sequence of pIN-III-ompA2. The site-specific mutagenesiswas carried out as disclosed by Zoller and Smith (cited above). Briefly,the XbaI/BamHI fragment of pIN-III-ompA2 containing the coding sequencefor the ompA signal peptide (see FIG. 1 in Ghrayeb et al., cited above)was purified, mixed with purified XbaI/BamHI digested replicating form(RF) of M13mp19, ligated, transfected into E. coli K-12 JM101, andplated. A clear plaque in the presence of IPTG and X-gal was selected,propagated, and single stranded DNAs were prepared, e.g. according tothe procedures disclosed by Messing, in Method in Enzymology, Vol. 101(Academic Press, New York, 1983). Separately, the followingoligonucleotide primer (23-mer) containing the indicated basesubstitutions (boxed) was synthesized and phosphorylated. ##STR1## Thissequence introduces a second HindIII site in the signal peptide codingregion of the mutated pIN-III-ompA2. The oligonucleotide primer wasannealed to the M13mp19 RF containing the XbaI/BamHI fragment ofpIN-III-ompA2, and treated with DNA polymerase in the presence ofappropriate concentrations of nucleoside triphosphates. The resulting RFwere used to transfect JM101 E. coli, and mutant-containing plaques werescreened by a labeled oligonucleotide probe. The sequence of theselected RF was confirmed by dideoxy sequencing using a universal M13primer. The selected RF was propagated, isolated, digested with XbaI andBamHI, and the purified XbaI/BamHI fragment was inserted into anXbaI/BamHI digested pIN-III-ompA2. To form pIN-III-ompA2(2), the mutantpIN-III-ompA2 was propagated, purified, digested with HindIII and BamHI,and mixed with the BamHI/EcoRV fragment of pcD-125 in a standardligation solution containing 0.1 micromolar of the following syntheticlinker:

    ______________________________________                                        A     GCT      CAC     AAG      TGC   GAT                                                    GTG     TTC      ACG   CTA                                     ______________________________________                                    

E. coli strain Ab1899 was infected by the pIN-III-ompA2(2) plasmid andtransformants were selected by colony hybridization using a ³² P-labeledIL-4 cDNA probe. IL-4 extracts, prepared as described above, exhibitedTCGF activity comparable to supernatants of pcD-125 COS7 cells.

B. TRPC11

The TRPC11 vector was constructed by ligating a synthetic consensus RBSfragment to ClaI linkers (ATGCAT) and by cloning the resulting fragmentsinto ClaI restricted pMT11hc (which had been previously modified tocontain the ClaI site). pMT11hc is a small (2.3 kilobase) high copy,AMP^(R), TET^(S) derivative of pBR322 that bears the πVX plasmid(described by Maniatis et al., cited above) EcoRI-HindIII polylinkerregion. It was modified to contain the ClaI site by restricting pMT11hcwith EcoRI and BamHI, filling in the resulting sticky ends and ligatingwith ClaI linker (CATCGATG), thereby restoring the EcoRI and BamHI sitesand replacing the SmaI site with a ClaI site.

One transformant from the TRPC11 construction had a tandem RBS sequenceflanked by ClaI sites. One of the ClaI sites and part of the second copyof the RBS sequence were removed by digesting this plasmid with PstI,treating with Bal31 nuclease, restricting with EcoRI and treating withT4 DNA polymerase in the presence of all four deoxynucleotidetriphosphates. The resulting 30-40 bp fragments were recovered via PAGEand cloned into SmaI restricted pUC12. A 248 bp E. coli trpP-bearingEcoRI fragment derived from pKC101 (described by Nichols et al. inMethods in Enzymology, Vol. 101, pg. 155 (Acedemic Press, N.Y. 1983))was then cloned into the EcoRI site to complete the TRPC11 construction,which is illustrated in FIG. 12.

TRPC11 was employed as a vector for human IL-4 cDNA by first digestingit with ClaI and Bam HI, purifying it, and then mixing it with theEcoRV/BamHI fragment of pcD-125 in a standard ligation solutioncontaining 0.1 micromolar of the following synthetic linker:

    ______________________________________                                        TCG     ATG          CAC   AAG     TGC   GAT                                          AC           GTG   TTC     ACG   CTA                                  ______________________________________                                         The insert-containing vector was selected as described above and     propagated in E. coli K-12 strain JM101. IL-4 was extracted as follows.     JM101 cells were sonicated in their culture medium and centrifuged. The     pellet was resuspended in 4M guanidine and 2 mM dithiothreitol, and again     centrifuged. The supernatant was tested for biological activity and found     to exhibit TCGF activity comparable to that of supernatants of pcD-125     transfected COS7 cells.

EXAMPLE V Preparation of Bovine IL-4 cDNAs Via Mouse and Human IL-4 cDNAProbes to a Bovine Helper T Cell cDNA Library and Transient Expressionin COS 7 Monkey Cells

cDNA clones coding for IL-4 are isolated from cDNA libraries constructedfrom induced bovine peripheral blood lymphocytes (PBLs) by way of acombined mouse and human cDNA probes. Alternative sources of bovinecDNAs include several bovine cell lines maintained in the ATCC's NBLanimal line collection. Procedures are substantially identical to thosedescribed in Example II. Cells are harvested about 10 hours afterinduction by Con A. mRNA extraction and cDNA library construction arecarried out as in Example II.

The mouse and human cDNA probes can be used together as a mixture orsequentially to detect bovine IL-4 cDNAs. As in Example II, the PstIfragment is isolated from the mouse pcD-2A-E3 cDNA clone. Likewise thePstI fragment is isolated from the human pcD-125 cDNA clone. Severalother fragments are also available to construct probes from. Eithertogether or separately the isolated PstI fragments are labeled by nicktranslation (about 1×10⁸ cpm/microgram) and are used to probenitrocellulose filters containing plasmid DNA preparations from 10pools, each representing about 1000 clones of the induced PBL cDNAlibrary. Filter hybridization is carried out as in Example II. Positivescoring clones are identified and propagated.

EXAMPLE VI Expression of Native Human IL-4 and Muteins Δ¹⁻⁴ and IS⁰(Gly-Asn-Phe-Val-His-Gly) in Saccharomyces cerevisiae

Native human IL-4 cDNA and two mutants thereof were cloned into theplasmid pMF-alpha8 and expressed in the yeast Saccharomyces cerevisiae.The construction and application of pMF-alpha8 for expressing non-yeastproteins is described fully in Miyajima et al., Gene, Vol. 37, pgs.155-161 (1985); and Miyajima et al., EMBO Journal, Vol. 5, pgs.1193-1197 (1986), both of which are incorporated by reference.pMF-alpha8 is deposited with the American Type Culture Collection(Rockville, MD) under accession number 40140, and a map of the plasmidis illustrated in FIG. 13A (designations in the figure are defined fullyin Miyajima et al., Gene, cited above).

A. Human IL-4 Mutein Δ¹⁻⁴.

Plasmid pcD-125 was isolated and digested with EcoRV and BamHI. TheEcoRV/BamHI fragment containing the human IL-4 cDNA was isolated,treated with DNA polymerase I (Klenow fragment) to fill in the BamHIcut, and kinased (i.e. phosphorylated). pMF-alpha8 was digested withStuI and combined with the kinased EcoRV/BamHI fragment of pcD-125 in astandard ligation solution to form plasmid phIL-4-2. phIL-4-2 was usedto transform S. cerevisiae 20B-12 (MATalpha trp1-289 pep4-3), which wasobtained from the Yeast Genetic Stock Center, University of California,Berkeley. Yeast cells were grown in synthetic medium containing 0.67%Yeast Nitrogen Base without amino acids, 2% glucose, and 0.5% Casaminoacids (Difco). The yeast cells were transformed with the plasmids by thelithium acetate method of Ito et al., J. Bacteriol., Vol. 153, pgs.163-168 (1983), and transformants were selected in synthetic mediumlacking tryptophan. Supernatant of a transformant culture was tested forTCGF activity. FIG. 13B (curve D) illustrates the TCGF activity ofseveral dilutions of the supernatant from phIL-4-2 transformed yeastcells in comparison with other factors (Curve A--human IL-2, CurveB--supernatant from pcD-125 transfected COS 7 cells, and CurveC--supernatants from phIL-4-1 transformed yeast cells). Curve Eillustrates the TCGF activity of supernatant from yeast that had beentransformed with pMF-alpha8 lacking the IL-4 cDNA insert, i.e. the"mock" transformant.

B. Human IL-4 Mutein IS⁰ (Gly-Asn-Phe-Val-His-Gly)

The pMF-alpha8 insert for expression of mutein IL⁰(Gly-Asn-Phe-Val-His-Gly) was prepared exactly as for mutein Δ¹⁻⁴,except that the NaeI/BamHI fragment from pcD-125 was used. The resultingplasmid was designated phIL-4-1. Several dilutions of supernatant fromphIL-4-1 transformed yeast cells were tested for TCGF activity. Theresults are illustrated by Curve C of FIG. 13B. The supernatants werealso tested for BCGF activity on both anti-IgM and SAC activated Blymphocytes. The assays were performed as described above, and theresults are given in Table VI.

                  TABLE VI                                                        ______________________________________                                        BCGF Activity of Supernatants of phIL-4-3                                     Transformed Yeast Cells                                                       % (vol/vol) [.sup.3 H] Thymidine Incorporation (cpm)                          of                         Anti-IgM                                           supernatants                                                                              SAC activated  Bead Activated                                     added       B Lymphocytes  B Lymphocytes                                      ______________________________________                                        .0          3633 ± 1239 641 ± 69                                        0.09        7610 ± 310  13221 ± 472                                     0.19        9235 ± 181  --                                                 0.39        10639 ± 786 16681 ± 310                                     0.78        10372 ± 572 18090 ± 1248                                    1.56        9905 ± 328  17631 ± 1216                                    3.12        11354 ± 836 18766 ± 1179                                    6.25        10481 ± 541 19810 ± 1349                                    12.5        9641 ± 30   18136 ± 1126                                    25.         8253 ± 857  14750 ± 1125                                    ______________________________________                                    

C. Expression of Native Human IL-4 in Yeast

cDNA coding for native human IL-4 was cloned into pMF-alpha8 by firstinserting bases upstream of the N-terminal His codon to form a KpnIrestriction site. After cleavage by KpnI and treatment by DNApolymeraseI, the blunt ended IL-4 cDNA was inserted into the StuI siteof pMF-alpha8. The KpnI site was formed by use of standard site-specificmutagenesis. Briefly, pcD-125 was digested with BamHI, the fragmentcontaining the entire human IL-4 cDNA was isolated, and inserted intothe BamHI site of M13mp8. Single stranded M13mp8 containing the insertwas isolated and hybridized to the following synthetic oligonucleotidewhich served as a primer: ##STR2## The inserted nucleotides are boxed.The plasmid containing the mutated IL-4 cDNA was identified by anoligonucleotide probe, propagated, isolated, and treated with KpnI andBamHI. The KpnI/BamHI fragment was isolated, treated with DNA polymeraseI (Klenow fragment) to generate blunt ends, kinased, and ligated withStuI digested pMF-alpha8. Yeast was transformed by the resultingpMF-alpha8 plasmids, designated phIL-4-3, as described above, andsupernatants were tested for TCGF activity. The supernatants displayedTCGF activity comparable to that observed for supernatants of phIL-4-1transformed yeast.

EXAMPLE VII Construction and Expression of a Synthetic Human IL-4 Genein E. coli

A synthetic human IL-4 gene is constructed which substantially comprisesbacterial preferred codons and which includes a series of uniquerestriction endonuclease sites (referred to herein as "uniquerestriction sites") which permits rapid and convenient expression of awide variety of human IL-4 muteins. The nucleotide sequence of thesynthetic gene is illustrated in FIG. 6A. Unique restriction sites withrespect to plasmid pUC18 are indicated in FIG. 6B. Techniques forconstructing and expressing the synthetic gene of this example arestandard in the art of molecular biology, e.g. Sproat and Gait, NucleicAcids Research, Vol. 13, pgs. 2959-2977 (1985); Mullenbach et al., J.Biol. Chem., Vol. 261, pgs. 719-722 (1986); Ferretti et al., Proc. Natl.Acad. Sci., Vol. 83, pgs. 599-603 (1986); Wells et al., Gene, Vol. 34,pgs. 315-323 (1985); and Estell et al., Science, Vol. 233, pgs. 659-663(1986). Sproat and Gait (cited above) and Ferretti et al. (cited above)are incorporated by reference as guides for applying the technique ofgene synthesis. Briefly, the synthetic human IL-4 gene is assembled froma plurality of chemically synthesized double stranded DNA fragments.Base sequences of the synthetic gene are selected so that the assembledsynthetic gene contains a series of unique restriction sites.

The series of unique restriction sites defines a series of segmentswhich can be readily excised and replaced with segments having alteredbase sequences. The synthetic fragments are inserted either directly orafter ligation with other fragments into a suitable vector, such as apUC plasmid, or the like. The above-mentioned segments roughlycorrespond to the "cassettes" of Wells et al. (cited above). Thesynthetic fragments are synthesized using standard techniques, e.g.Gait, Oligonucleotide Synthesis: A Practical Approach (IRL Press,Oxford, UK, 1984). Preferably an automated synthesizer is employed, suchas an Applied Biosystems, Inc. (Foster City, CA) model 380A. pUCplasmids and like vectors are commercially available, e.g. Pharmacia-PL,or Boehringer-Mannheim. Cloning and expression can be carried out instandard bacterial systems, for example E. coli K-12 strain JM101,JM103, or the like, described by Viera and Messing, in Gene, Vol. 19,pgs. 259-268 (1982).

Restriction endonuclease digestions and ligase reactions are performedusing standard protocols, e.g. Maniatis et al., Molecular Cloning: ALaboratory Manual (Cold Spring Harbor Laboratory, N.Y., 1982).

The alkaline method (Maniatis et al., cited above) is used for smallscale plasmid preparations. For large scale preparations a modificationof the alkaline method is used in which an equal volume of isopropanolis used to precipitate nucleic acids from the cleared lysate.Precipitation with cold 2.5M ammonium acetate is used to remove RNAprior to cesium chloride equilibrium density centrifugation anddetection with ethidium bromide.

For filter hybridizations Whatman 540 filter circles are used to liftcolonies which are then lysed and fixed by successive treatments with0.5M NaOH, 1.5M NaCl; 1M Tris.HCl pH8.0, 1.5M NaCl (2 min each); andheating at 80° C. (30 min). Hybridizations are in 6×SSPE, 20% formamide,0.1% sodium dodecylsulphate (SDS), 100 μg/ml E. coli tRNA, 100 μg/mlCoomassie Brilliant Blue G-250 (Biorad) at 42° C. for 6 hrs using ³²P-labelled (kinased) synthetic DNAs. (20×SSPE is prepared by dissolving174 g of NaCl, 27.6 g of NaH₂ PO₄.H₂ O, and 7.4 g of EDTA in 800 ml ofH₂ O. pH is adjusted to 7.4 with NaOH, volume is adjusted to 1 liter,and sterilized by autoclaving).

Filters are washed twice (15 min, room temperature) with 1×SSPE, 0.1%SDS. After autoradiography (Fuji RX film), positive colonies are locatedby aligning the regrown colonies with the blue-stained colonies on thefilters.

DNA is sequenced by either the chemical degradation method of Maxam andGilbert, Methods in Enzymology, Vol. 65, pg. 499 (1980), or by thedideoxy method, Sanger et al. Proc. Natl. Acad. Sci., Vol. 74, pg. 5463(1977). Templates for the dideoxy reactions are either single strandedDNAs of relevant regions recloned into M13mp vectors, e.g. Messing etal. Nucleic Acids Res., Vol. 9, pg. 309 (1981), or double-Stranded DNAprepared by the minialkaline method and denatured with 0.2M NaOH (5 min,room temperature) and precipitated from 0.2M NaOH, 1.43M ammoniumacetate by the addition of 2 volumes of ethanol. Dideoxy reactions aredone at 42° C.

DNA is synthesized by phosphoramidite chemistry using Applied Biosystems380A synthesizers. Synthesis, deprotection, cleavage and purification(7M urea PAGE, elution, DEAE-cellulose chromotography) are done asdescribed in the 380A synthesizer manual. Complementary strands ofsynthetic DNAs to be cloned (400ng each) are mixed and phosphorylatedwith polynucleotide kinase in a reaction volume of 50 μl. This DNA isligated with 1 μg of vector DNA digested with appropriate restrictionenzymes, and ligations are in a volume of 50 μl at room temperature for4 to 12 hours. Conditions for phosphorylation, restriction enzymedigestions, polymerase reactions, and ligation have been described(Maniatis et al., cited above). Colonies are scored for lacZ⁺ (whendesired) by plating on L agar supplemented with ampicillin,isopropyl-1-thio-beta-D-galactoside (IPTG) (0.4 mM) and5-bromo-4-chloro-3-indolyl-beta-D-galactopyranoside (x-gal) (40 μg/ml).

The TAC-RBS vector is constructed by filling-in with DNA polymerase thesingle BamHI site of the tacP-bearing plasmid pDR540 (Pharmacia). Thiswas then ligated to unphosphorylated synthetic oligonucleotides(Pharmacia) which form a double-stranded fragment encoding a concensusribosome binding site (RBS, GTAAGGAGGTTTAAC). After ligation, themixture was phosphorylated and religated with the SstI linkerATGAGCTCAT. This complex was then cleaved with SstI and EcoRI, and the173 bp fragment isolated via polyacrylamide gel electrophoresis (PAGE)and cloned into EcoRI-SstI restricted pUC18 (Pharmacia) (as describedbelow). The sequence of the RBS-ATG-polylinker regions of the finalconstruction (called TAC-RBS) is shown in FIG. 8.

The synthetic human IL-4 gene is assembled into a pUC18 plasmid in sixsteps. At each step inserts free of deletions and/or inserts can bedetected after cloning by maintaining the lacZ(α) gene of pUC18 in framewith the ATG start codon inserted in step 1. Clones containing deletionand/or insertion changes can be filtered out by scoring for bluecolonies on L-ampicillin plates containing x-gal and IPTG.Alternatively, at each step sequences of inserts can be readilyconfirmed using a universal sequencing primer on small scale plasmid DNApreparations, e.g. available from Boehringer Mannheim.

In step 1 the TAC-RBS vector is digested with SstI, treated with T4 DNApolymerase (whose 3' exonuclease activity digests the 3' protrudingstrands of the SstI cuts to form blunt end fragments), and afterdeactivation of T4 DNA polymerase, treated with EcoRI to form a 173 basepair (bp) fragment containing the TAC-RBS region and having a blunt endat the ATG start codon and the EcoRI cut at the opposite end. Finally,the 173 bp TAC-RBS fragment is isolated.

In step 2 the isolated TAC-RBS fragment of step 1 is mixed withEcoRI/SstI digested plasmid pUC18 and synthetic fragment 1A/B, which asshown in FIG. 7A has a blunt end at its upstream terminus and astaggered end corresponding to an SstI cut at its downstream terminus.The fragments are ligated to form the pUC18 of step 2.

In step 3 synthetic fragments 2A/B and 3A/B (illustrated in FIGS. 7B and7C) are mixed with SstI/BamHI digested pUC18 of step 2 (afteramplification and purification) and ligated to form pUC18 of step 3.Note that the downstream terminus of fragment 2A/B contains extra baseswhich form the BamHI staggered end. These extra bases are cleaved instep 4. Also fragments 2A/B and 3A/B have complementary 9 residue singlestranded ends which anneal upon mixture, leaving the upstream SstI cutof 2A/B and the downstream BamHI cut of 3A/B to ligate to the pUC18.

In step 4 MluI-XbaI digested pUC18 of step 3 (after amplification andpurification) is repurified, mixed with synthetic fragment 4A/B (FIG.7D), and ligated to form pUC18 of step 4.

In step 5 XbaI/SalI digested pUC18 of step 4 (after amplification andpurification) is mixed with synthetic fragment of 5A/B (FIG. 7E) andligated to form the pUC18 of step 5.

In step 6 SalI/HindIII digested pUC18 of step 5 (after amplification andpurification) is mixed with synthetic fragment 6A/B (FIG. 7F) andligated to form the final construction.

FIG. 6B is a cleavage map of the unique restriction sites present in thepUC18 construction just described. When the disclosed synthetic humanIL-4 gene is used as an insert of pUC18 each pair of unique restrictionsites defines a segment which can be readily excised and replaced withaltered synthetic segments. The set of unique restriction sites includesEcoRI, HpaI, SacI (SstI), EcoRV, PstI, MluI, BclI, XbaI, NaeI, SalI,XhoI, and HindIII.

The pUC18 containing the synthetic IL-4 gene is inserted in E. coli K-12strain JM101. After culturing, protein is extracted from the JM101 cellsand dilutions of the extracts are tested for biological activity.

EXAMPLE VIII Construction and Expression of Human IL-4 Mutein Ile⁵² inE. coli

Leu at position 52 (relative to the N-terminus of the native human IL-4)is changed to Ile to form human IL-4 mutein Ile⁵². The pUC18 plasmid ofExample VII containing the synthetic human IL-4 gene of FIG. 6A isdigested with PstI and MluI and purified. The above purified pUC18 ismixed with the synthetic double stranded fragment illustrated below andligated. The altered part of the base sequence is boxed. The resultingpUC18 is transfected into E. coli K-12 strain JM101, or the like, andexpressed.

    __________________________________________________________________________    PstI/MluI Replacement Fragment For                                            Generating Human Il-4 Mutean Ile.sup.52                                       __________________________________________________________________________         GA  GCT  GCT ACC  GTT ATC  CGT                                           ACG  TCT CGA  CGA TGG  CAA TAG  GCA                                           CAG  TTC TAC  TCT CAC  CAC GAA  AAA                                           GTC  AAG ATG  AGA GTG  GTG CTT  TTT                                           GAC  A                                                                        CTG  TGC GC                                                                   __________________________________________________________________________

After culturing, protein is extracted from the JM101 cells usingstandard techniques, and dilutions of the extracts are tested forbiological activity.

EXAMPLE IX Construction and Expression of Human IL-4 Mutein (Ile⁵²,Asp¹¹¹)

The modified pUC18 plasmid of Example VIII (containing the Ile⁵² codingsequence) is digested with SalI and XhoI, and the large fragment isisolated. The isolated fragment is mixed with the synthetic doublestranded fragment illustrated below in a standard ligation solution. Thealtered part of the sequence is boxed. The resulting plasmid istransfected into E. coli K-12 strain JM101, or the like, and expressed.

    ______________________________________                                        TCG   ACT    CTG     GAA   GAC   TTC   C                                            GA     GAC     CTT   CTG   AAG   GAG   CT                               ______________________________________                                    

After culturing, protein is extracted from the JM101 cells usingstandard techniques, and dilutions of the extracts are tested forbiological activity.

EXAMPLE X Sequence of Human IL-4 Purified from Transfection Supernatants

Human IL-4 was purified from culture supernatants of cells transientlytransfected with vectors containing human IL-4 cDNA. The sequence of thesecreted native human IL-4 was determined from the purified material.

A. Biological Assay for Purification

TCGF activity was used to assay human IL-4 during the separationprocedures. The assay was substantially the same as that described inExample II. Briefly, blood from a healthy donor was drawn into aheparinized tube and layered onto Ficoll-Hypaque; e.g., 5 ml of bloodper 3 ml Ficoll-Hypaque in a 15 ml centrifuge tube. After centrifugationat 3000×g for 20 minutes, cells at the interface were aspirated anddiluted in a growth medium consisting of RPMI 1640 containing 10% fetalcalf serum, 50 micromolar 2-mercaptoethanol, 20 microgram/mlphytohemagglutinin (PHA), and recombinant human IL-2. After 5-10 days ofincubation at 37° C, the PHA-stimulated peripheral blood lymphocytes(PBLs) were washed and used in 2 day colorimetric assays, Mossmann, J.Immunol. Methods, Vol. 65, pgs. 55-63 (1983). Serial two fold dilutionsof the IL-4 standard (supernatants from either pcD-125 or pEBT-178transfected COS 7 cells) or the fraction to be tested were performed in96 well trays utilizing the growth medium described above to yield afinal volume of 50 microliters/well. 50 microliters of the PHAstimulated PBLs at about 4-8×10⁶ cells/ml were added to each well andthe trays were incubated at 37° C. for 2 days. Cell growth was thenmeasured according to Mosmann (cited above).

Units of human IL-4 TCGF activity are defined with respect tosupernatants of either pcD-125 transfected COS 7 cells (Example II) orpEBV-178 transfected COS 7 cells (Example III).

For purification, units are based on the activity of pcD-125transfection supernatants, which are produced as follows. About 1×10⁶COS 7 cells are seeded onto 100 mm tissue culture plates containingDulbecco's Modified Eagle's medium (DME), 10% fetal calf serum, and 4 mML-glutamine. About 24 hours after seeding, the medium is aspirated fromthe plates and the cells are washed twice with serum free buffered (50mM Tris) DME. To each plate is added 4 ml serum free buffered DME (with4 mM L-glutamine), 80 microliters DEAE-dextran, and 5 micrograms ofpcD-125 DNA. The cells are incubated in this mixture for 4 hours at 30°C., after which the mixture is aspirated off and the cells are washedonce with serum free buffered DME. After washing, 5 ml of DME with 4 mML-glutamine, 100 micromolar Chloroquine, and 2% fetal calf serum isadded to each plate, the cells are incubated for 3 hours, and then twicewashed with serum free buffered DME. Next, 5 ml DME with 4 mML-glutamine and 4% fetal calf serum is added and the cells are incubatedat 37° C. for 24 hours. Afterwards the cells are washed 1-3 times withDME or PBS, 5 ml serum free DME (with 4 mM L-glutamine) is added, andthe cells are incubated at 37° C. until culture supernatants areharvested 5 days later.

One unit, as used herein, is the amount of factor which in one well (0.1ml) stimulates 50% maximal proliferation of 2×10⁴ PHA stimulated PBLsover a 48 hour period.

B. Purification

Purification was accomplished by a sequential application of cationexchange chromatography, gel filtration and reverse-phase high pressureliquid chromatography. All operations were performed at 4° C.

After removing the COS 7 cells by centrifugation, the supernatant wasconcentrated about 10 fold by ultrafiltration and stored at -80° C.until further processed. IL-4 titers were determined by assaying for theability of the protein to stimulate proliferation ofphytohemagglutinin-induced human peripheral blood lymphocytes, i.e. byTCGF activity using the standard assay described above.

Concentrated COS 7 supernatant, having TCGF activity of about 10⁴ -10⁶units/ml and a protein content of about 15-20 mg/ml, is dialyzed against2 changes of 50 mM sodium HEPES, pH 7.0 over a 24 hour period (eachchange being approximately 10-15 times the volume of one concentrate).The dialysate was applied to a column (1×2.5 cm) of S-Sepharose (flowrate: 0.2 ml/min) pre-equilibrated with 50 mM sodium HEPES, pH 7.0. Thecolumn were washed with 15 column volumes of equilibrating bufferfollowed by elution with 20 column volumes of a linear sodium chloridegradient extending from 0 to 0.5 M sodium chloride in 50 mM sodiumHEPES, pH 7.0. The gradient was terminated with an isocratic elutionconsisting of 5 column volumes of 50 mM sodium HEPES, 0.5 M NaCl, pH7.0. 1.5 ml and 1.8 ml fractions were collected from respective batches.IL-4 titers were found for both chromatographies to elute between 300 mMand 500 mM sodium chloride.

The fractions from the S-Sepharose columns containing IL-4 titers werecombined for total separate volumes of 9.0 and 10.8 ml. Both volumeswere concentrated to 1.9 ml by ultrafiltration using an Amicon YM5membrane (molecular weight cut-off: 5000). The recovery of protein fromthis step was about 80%. The concentrated IL-4 solution was applied to aSephadex G-100 column (1.1×58 cm) pre-equilibrated in 50 mM HEPES, 0.4 MNaCl, pH 7.0 and the column was eluted with the same buffer at 0.15ml/min. A total of 50 fractions (1.0 ml/fraction) was collected andanalyzed for IL-4 titers. A peak in biological activity was observed atan apparent molecular weight of 22,000 daltons. The Sephadex G-100 wascalibrated for apparent molecular determination with bovine serumalbumin (65,000 daltons), carbonic anhydrase (30,000 daltons) andcytochrome C (11,700 daltons).

A fraction from the Sephadex G-100 column containing IL-4 activity wasconcentrated 3-4 fold in vacuo and was injected onto a Vydac C-4 guardcolumn (4.6×20 mm). A linear gradient of 0 to 72% (v/v) acetonitrile in0.1% (v/v) trifluoroacetic acid (TFA) was produced in 15 minutes at acolumn temperature of 35° and a flow rate of 1.0 ml/min. Three peaksresulted that were detected at 214 nm with retention times of 7, 8.2 and8.7 min. (peaks 1, 2, and 3 of FIG. 10, respectively). A 40 microliteraliquot of peak 2 (8.2 min. elution time) was lyophilized andredissolved in minimal essential medium containing 10% fetal calf serum.This solution showed a positive TCGF response. A 300 microliter aliquotof peak 2 was evaporated to dryness and redissolved in 200 ul of 0.1%(w/v) sodium dodecyl sulfate (SDS). A 2 ul aliquot was diluted in 200 ulof 1% (v/v) TFA and rechromatographed. The HPLC of this sampledemonstrated a single peak at 215 nm. Peak 2 material indicated anactivity of about 7×10⁸ units/mg.

C. Amino Acid Sequence Analysis

Amino Acid sequence determination was performed by automated gas-phaseEdman degradation (Hewick, R. M., Hunkapillar, M. W., Hood, L. E. andDryer, W. J. (1981) J. Biol. Chem. 256: 7990.) employing an AppliedBiosystems microsequenator. A 90 microliter aliquot of the peak 2 HPLCfraction, dissolved in 0.1% SDS as described above was applied to theglass fiber filter cartridge in the presence of Polybrene. Amino acidsequence information was obtained up to the 35th residue. The N-terminalsequence was determined as follows

    ______________________________________                                        His--Lys--.sub.-- --Asp--Ile--Thr--Leu--Gln--Glu--                            Ile--Ile--Lys--Thr--Leu--Asn--Ser--Leu--Thr--                                 Glu--Gln--Lys--Thr--Leu--.sub.-- --Thr--Glu--Leu--                            .sub.-- --Val--Thr--Asp--Ile--Phe--Ala--Ala                                   ______________________________________                                    

wherein the blanks indicate the lack of an identifiable amino acid.

Blanks in the amino-terminus at positions 3 and 23 were consistent withthe presence of cysteine, which can not be detected in this system. Theblank at position 28, corresponding to a threonine in the cDNA-predictedsequence, may have been due either to the variability ofphenylthiohydantoin-threonine detection or to the presence of O-linkedglycosylation or esterification.

A 100 microliter aliquot of the HPLC fraction on which theamino-terminal sequence was performed was evaporated to dryness andredissolved in 70% formic acid. A 50-fold molar excess of cyanogenbromide was added and the solution was allowed to stand at roomtemperature for 2.5 hours. The cleaved protein was sequenced on theApplied Biosystems gas-phase sequenator, as described above. Twosequences were identifiable:

    Arg-Glu-Lys-Tyr-Ser-Lys                                    (1)

    His-Lys-.sub.-- -Asp-Ile-Thr                               (2)

Sequence (1) is identical to the sequence predicted from the cDNA tohave been released following cyanogen bromide cleavage of the methionineresidue at position 120. The last 2 residues of the C-terminus may havebeen present but may not have been detectable due to insufficientsample. Sequence (2) is identical to the amino-terminal sequenceobtained for the native IL-4 protein, as described above. The relativeamounts of amino-terminal and carboxyl-terminalphenylthiohydantoin-amino acids released suggested equimolar amounts ofboth sequences in the sample. This result supports the conclusion thatthe protein sample that was sequenced contained predominantly a singlepolypeptide chain with amino and carboxyl termini predicted from thecDNA sequence of human IL-4.

EXAMPLE XI Construction and Expression of Human IL-4 Mutein (Ile⁵², Δ⁷¹,IS⁹⁴ (Ala))

The modified pUC18 plasmid of Example VIII (containing the Ile⁵² codingsequence) is digested with MluI and BclI, and the large fragment isisolated. The isolated fragment is mixed with the synthetic doublestranded fragment illustrated below in a standard ligation solution. Theresulting plasmid is transfected into E. coli K-12 JM101, or the like,and propagated.

    __________________________________________________________________________    CG  CGT TGT CTC GGC GCC ACT                                                       A   ACA GAG CCG CGG TGA                                                   GCG CAG TTC CAC CGT CAC AAA GAG CT                                            CGC GTC AAG GTG GCA GTG TTT GTC GAC TAG                                       ↑                                                                       Deletion                                                                      __________________________________________________________________________

The modified plasmid is isolated and digested with XbaI and NaeI, andthe large fragment is isolated. The isolated fragment is mixed with thesynthetic double stranded fragment illustrated below in a standardligation solution. The added codon is boxed. The resulting plasmid istransfected into E. coli K-12 strain JM101, or the like, and expressed.

    ______________________________________                                        CTA    GAC     CGT     AAC    CTG   TGG   GGC                                        TG      GCA     TTG    GAC   ACC   CCG                                 CTG    GCC     GCC                                                            GAC    CGG     CGG                                                            ______________________________________                                    

After culturing, protein is extracted from the JM101 cells usingstandard techniques, and dilutions of the extracts are tested forbiological activity.

EXAMPLE XII Induction of DR Antigens on Cells from a Patient Sufferingfrom Bare Lymphocyte Syndrome

Bare lymphocyte syndrome is characterized by the lack of expression ofclass I and/or class II HLA antigens on cell surfaces, and is frequentlyassociated with severe immunodeficiency, e.g. Touraine, Lancet, pgs.319-321 (Feb. 7, 1981); Touraine and Bethel, Human Immunology, Vol. 2,pgs. 147-153 (1981); and Sullivan et al., J. Clin. Invest., Vol. 76,pgs. 75-79 (1985). It was discovered that human IL-4 was capable ofinducing the expression of the class II DR antigen on the surfaces ofcells derived from a patient suffering from bare lymphocyte syndrome.

Peripheral blood lymphocytes (PBLs) were obtained from a patientsuffering from non-expression of HLA class II antigens. B cells werepurified from the PBLs essentially as described above, and a B cell line(designated UD31) was established by transformation with Epstein-Barrvirus (EBV). The EBV-transformed cells were cultured for 48 hours inYssel's defined medium (described above) with 2% fetal calf serum and a5% (v/v) concentration of supernatant from pcD-125 transfected COS 7cells. The cells were harvested, fixed, stained with fluorescentlylabeled anti-DR monoclonal antibody (e.g. Becton Dickinson, L243), andanalyzed flow cytometrically. FIG. 9 illustrates histograms of cellfrequency versus fluorescence intensity for a control population of theEBV-transformed cells harvested prior to IL-4 treatment (Curve A), andfor the population of EBV-transformed cells after IL-4 treatment (CurveB).

The descriptions of the foregoing embodiments of the invention have beenpresented for purpose of illustration and description. They are notintended to be exhaustive or to limit the invention to the precise formsdisclosed, and obviously many modifications and variations are possiblein light of the above teaching. The embodiments were chosen anddescribed in order to best explain the principles of the invention andits practical application to thereby enable others skilled in the art tobest utilize the invention in various embodiments and with variousmodifications as are suited to the particular use contemplated. It isintended that the scope of the invention be defined by the claimsappended hereto.

Applicants have deposited cDNA clones pcD-2A-E3, pcD-46 (pcD-2F1-13),pcD-125, and yeast vector pMF-alpha8 with the American Type CultureCollection, Rockville, MD, USA (ATCC), under accession numbers 53330,53337, 67029, and 40140, respectively. These deposits were made underconditions as provided under ATCC's agreement for Culture Deposit forPatent Purposes, which assures that these deposits will be madeavailable to the US Commissioner of Patents and Trademarks pursuant to35 USC 122 and 37 CFR 1.14, and will be made available to the publicupon issue of a U.S. patent, which requires this deposit to bemaintained. Availability of the deposited strains is not to be construedas a license to practice the invention in contravention of the rightsgranted under the authority of any government in accordance with itspatent laws.

We claim:
 1. Human interleukin-4 substantially free from any other proteinaceous material.
 2. The human interleukin-4 of claim 1 having at least 9×10⁷ units/mg of TCGF activity.
 3. The compound of claim 1 comprising a polypeptide selected from the group consisting of 1-fold substituted polypeptides having a sequence of amino acids defined by the formula:

    ______________________________________                                         X(His)--X(Lys)--X(Cys)--X(Asp)--X(Ile)--X(Thr)--                               X(Leu)--X(Gln)--X(Glu)--X(Ile)--X(Ile)--X(Lys)--                               X(Thr)--X(Leu)--X(Asn)--X(Ser)--X(Leu)--X(Thr)--                               X(Glu)--X(Gln)--X(Lys)--X(Thr)--X(Leu)--X(Cys)--                               X(Thr)--X(Glu)--X(Leu)--X(Thr)--X(Val)--X(Thr)--                               X(Asp)--X(Ile)--X(Phe)--X(Ala)--X(Ala)--X(Ser)--                               X(Lys)--X(Asn)--X(Thr)--X(Thr)--X(Glu)--X(Lys)--                               X(Glu)--X(Thr)--X(Phe)--X(Cys)--X(Arg)--X(Ala)--                               X(Ala)--X(Thr)--X(Val)--X(Leu)--X(Arg)--X(Gln)--                               X(Phe)--X(Tyr)--X(Ser)--X(His)--X(His)--X(Glu)--                               X(Lys)--X(Asp)--X(Thr)--X(Arg)--X(Cys)--X(Leu)--                               X(Gly)--X(Ala)--X(Thr)--X(Ala)--X(Gln)--X(Gln)--                               X(Phe)--X(His)--X(Arg)--X(His)--X(Lys)--X(Gln)--                               X(Leu)--X(Ile)--X(Arg)--X(Phe)--X(Leu)--X(Lys)--                               X(Arg)--X(Leu)--X(Asp)--X(Arg)--X(Asn)--X(Leu)--                               X(Trp)--X(Gly)--X(Leu)--X(Ala)--X(Gly)--X(Leu)--                               X(Asn)--X(Ser)--X(Cys)--X(Pro)--X(Val)--X(Lys)--                               X(Glu)--X(Ala)--X(Asn)--X(Gln)--X(Ser)--X(Thr)--                               X(Leu)--X(Glu)--X(Asn)--X(Phe)--X(Leu)--X(Glu)--                               X(Arg)--X(Leu)--X(Lys)--X(Thr)--X(Ile)--X(Met)--                               X(Arg)--X(Glu)--X(Lys)--X(Tyr)--X(Ser)--X(Lys)--                               X(Cys)--X(Ser)--X(Ser)                                                         ______________________________________                                    

wherein said polypeptide exhibits T cell growth factor activity and B cell growth factor activity, and wherein: X(Cys) is Cys; X(Trp) is Trp; X(Ser) represents the group consisting of Ser, Thr, Gly, and Asn; X(Arg) represents the group consisting of Arg, His, Gln, Lys, and Glu; X(Leu) represents the group consisting of Leu, Ile, Phe, Tyr, Met, and Val; X(Pro) represents the group consisting of Pro, Gly, Ala, and Thr; X(Thr) represents the group consisting of Thr, Pro, Ser, Ala, Gly, His, and Gln; X(Ala) represents the group consisting of Ala, Gly, Thr, and Pro; X(Val) represents the group consisting of Val, Met, Tyr, Phe, Ile, and Leu; X(Gly) represents the group consisting of Gly, Ala, Thr, Pro, and Ser; X(Ile) represents the group consisting of Ile, Met, Tyr, Phe, Val, and Leu; X(Phe) represents the group consisting of Phe, Trp, Met, Tyr, Ile, Val, and Leu; X(Tyr) represents the group consisting of Tyr, Trp, Met, Phe, Ile, Val, and Leu; X(His) represents the group consisting of His, Glu, Lys, Gln, Thr, and Arg; X(Gln) represents the group consisting of Gln, Glu, Lys, Asn, His, Thr, and Arg; X(Asn) represents the group consisting of Asn, Glu, Asp, Gln, and Ser; X(Lys) represents the group consisting of Lys, Glu, Gln, His, and Arg; X(Asp) represents the group consisting of Asp, Glu, and Asn; X(Glu) represents the group consisting of Glu, Asp, Lys, Asn, Gln, His, and Arg; and X(Met) represents the group consisting of Met, Phe, Ile, Val, Leu, and Tyr.
 4. The compound of claim 3 wherein:X(Ser) is Ser; X(Arg) represents the group consisting of Arg, His, and Lys; X(Leu) represents the group consisting of Leu, Ile, Phe, and Met; X(Pro) represents the group consisting of Pro and Ala; X(Thr) is Thr; X(Ala) represents the group consisting of Ala and Pro; X(Val) represents the group consisting of Val, Met, and Ile; X(Gly) is Gly; X(Ile) represents the group consisting of Ile, Met, Phe, Val, and Leu; X(Phe) represents the group consisting of Phe, Met, Tyr, Ile, and Leu; X(Tyr) represents the group consisting of Tyr and Phe; X(His) represents the group consisting of His, Gln, and Arg; X(Gln) represents the group consisting of Gln, Glu, and His; X(Asn) represents the group consisting of Asn and Asp; X(Lys) represents the group consisting of Lys and Arg; X(Asp) represents the group consisting of Asp and Asn; X(Glu) represents the group consisting of Glu and Gln; and X(Met) represents the group consisting of Met, Phe, Ile, Val, and Leu.
 5. The compound of claim 4 wherein:X(Arg) is Arg; X(Leu) represents the group consisting of Leu, Ile, and Met; X(Pro) is Pro; X(Ala) is Ala; X(Val) is Val; X(Ile) represents the group consisting of Ile, Met, and Leu; X(Phe) is Phe; X(Tyr) is Tyr; X(His) is His; X(Gln) is Gln; X(Asn) is Asn; X(Lys) is Lys; X(Asp) is Asp; X(Glu) is Glu; and X(Met) represents the group consisting of Met, Ile, and Leu.
 6. The compound of claim 5 wherein said glycosylated or unglycosylated polypeptide is defined by the formula:

    ______________________________________                                         His--Lys--Cys--Asp--Ile--Thr--Leu--Gln--Glu--Ile--                             Ile--Lys--Thr--Leu--Asn--Ser--Leu--Thr--Glu--Gln--                             Lys--Thr--Leu--Cys--Thr--Glu--Leu--Thr--Val--Thr--                             Asp--Ile--Phe--Ala--Ala--Ser--Lys--Asn--Thr--Thr--                             Glu--Lys--Glu--Thr--Phe--Cys--Arg--Ala--Ala--Thr--                             Val--Leu--Arg--Gln--Phe--Tyr--Ser--His--His--Glu--                             Lys--Asp--Thr--Arg--Cys--Leu--Gly--Ala--Thr--Ala--                             Gln--Gln--Phe--His--Arg--His--Lys--Gln--Leu--Ile--                             Arg--Phe--Leu--Lys--Arg--Leu--Asp--Arg--Asn--Leu--                             Trp--Gly--Leu--Ala--Gly--Leu--Asn--Ser--Cys--Pro--                             Val--Lys--Glu--Ala--Asn--Gln--Ser--Thr--Leu--Glu--                             Asn--Phe--Leu--Glu--Arg--Leu--Lys--Thr--Ile--Met--                             Arg--Glu--Lys--Tyr--Ser--Lys--Cys--Ser--Ser.                                   ______________________________________                                    


7. A human interleukin-4 mutein having the amino acid sequence:

    ______________________________________                                         Gly Asn Phe Val His Gly His Lys Cys Asp Ile Thr                                Leu Gln Glu Ile Ile Lys Thr Leu Asn Ser Leu Thr                                Glu Gln Lys Thr Leu Cys Thr Glu Leu Thr Val Thr                                Asp Ile Phe Ala Ala Ser Lys Asn Thr Thr Glu Lys                                Glu Thr Phe Cys Arg Ala Ala Thr Val Leu Arg Gln                                Phe Tyr Ser His His Glu Lys Asp Thr Arg Cys Leu                                Gly Ala Thr Ala Gln Gln Phe His Arg His Lys Gln                                Leu Ile Arg Phe Leu Lys Arg Leu Asp Arg Asn Leu                                Trp Gly Leu Ala Gly Leu Asn Ser Cys Pro Val Lys                                Glu Ala Asn Gln Ser Thr Leu Glu Asn Phe Leu Glu                                Arg Leu Lys Thr Ile Met Arg Glu Lys Tyr Ser Lys                                Cys Ser Ser.-                                                                  ______________________________________                                    


8. A human interleukin-4 mutein having the amino acid sequence:

    ______________________________________                                         Ala Glu Phe His Lys Cys Asp Ile Thr Leu Gln Glu                                Ile Ile Lys Thr Leu Asn Ser Leu Thr Glu Gln Lys                                Thr Leu Cys Thr Glu Leu Thr Val Thr Asp Ile Phe                                Ala Ala Ser Lys Asn Thr Thr Glu Lys Glu Thr Phe                                Cys Arg Ala Ala Thr Val Leu Arg Gln Phe Tyr Ser                                His His Glu Lys Asp Thr Arg Cys Leu Gly Ala Thr                                Ala Gln Gln Phe His Arg His Lys Gln Leu Ile Arg                                Phe Leu Lys Arg Leu Asp Arg Asn Leu Trp Gly Leu                                Ala Gly Leu Asn Ser Cys Pro Val Lys Glu Ala Asn                                Gln Ser Thr Leu Glu Asn Phe Leu Glu Arg Leu Lys                                Thr Ile Met Arg Glu Lys Tyr Ser Lys Cys Ser                                    Ser.-                                                                          ______________________________________                                    


9. A human interleukin-4 mutein having the amino acid sequence:

    ______________________________________                                         Ile Thr Leu Gln Glu Ile Ile Lys Thr Leu Asn Ser                                Leu Thr Glu Gln Lys Thr Leu Cys Thr Glu Leu Thr                                Val Thr Asp Ile Phe Ala Ala Ser Lys Asn Thr Thr                                Gly Lys Glu Thr Phe Cys Arg Ala Ala Thr Val Leu                                Arg Gln Phe Tyr Ser His His Glu Lys Asp Thr Arg                                Cys Leu Gly Ala Thr Ala Gln Gln Phe His Arg His                                Lys Gln Leu Ile Arg Phe Leu Lys Arg Leu Asp Arg                                Asn Leu Trp Gly Leu Ala Gly Leu Asn Ser Cys Pro                                Val Lys Glu Ala Asn Gln Ser Thr Leu Glu Asn Phe                                Leu Glu Arg Leu Lys Thr Ile Met Arg Glu Lys Tyr                                Ser Lys Cys Ser Ser.-                                                          ______________________________________                                     